Cross-linked elastomeric proteins in polar nonaqueous solvents and uses thereof

ABSTRACT

Disclosed herein are improved cross-linked resilin compositions and methods of making these improved compositions. These include new methods of cross-linking resilin compositions, use of polar nonaqueous solvents for adjusting material properties of cross-linked resilin solid compositions, and resilin foam compositions and methods of making the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/700,197, filed Jul. 18, 2018, the contents of which are incorporated by reference in their entirety.

BACKGROUND

Elastomeric proteins are polypeptides that exhibit viscoelastic mechanical properties, and include elastin, resilin, abductin, and octopus arterial elastomers. Resilin is a particularly interesting elastomeric protein because it dissipates very little energy during loading and unloading. Resilin is found in many insects, and the low energy dissipation enables the extraordinary ability of many insect species to jump or pivot their wings very efficiently. The unique properties of resilin make it an interesting elastomeric material that could have many industrial applications. What is needed, therefore, are new resilin compositions and methods of making the same that have desirable mechanical properties and are suitable for large-scale, efficient production.

Variations of natural resilins and resilin-like proteins (based on resilin sequences) have been recombinantly produced by a number of groups in E. coli cultures, and have been isolated by lysing the cells to extract recombinantly expressed proteins, and using affinity chromatography techniques to purify (Elvin et al., 2005; Charati et al.; 2009, McGann et al., 2013). The recombinantly produced resilin and resilin-like proteins have been cross-linked targeting the tyrosine residues that also form the cross-links in natural resilin (see, for example, Elvin et al., 2005; Qin et al., 2011). Recombinantly produced resilin has also been cross-linked targeting lysine residues (Li et al., 2011) or cysteine residues (McGann et al., 2013). Cross-linking methods have included enzymatically catalyzed cross-linking and photoactivated cross-linking. Enzymes for enzymatic catalysis of cross-linking can be hard to remove from the final composition, while photoactivated cross-linking may suffer from inefficiency due to only the surface being exposed to light, while also leaving impurities. The enzymatic impurities can degrade the resilin, while impurities from either enzymes or photocatalysts left in the final solid cross-linked composition can result in undesirable mechanical properties. What is needed, therefore, are new methods of cross-linking recombinant resilin proteins that do not leave behind degradation causing impurities and can be performed efficiently at a large scale.

SUMMARY OF THE INVENTION

Disclosed herein are improved cross-linked resilin compositions and methods of making these improved compositions. These include new methods of cross-linking resilin compositions and the use of polar nonaqueous solvents for adjusting material properties of cross-linked resilin solid compositions.

In some embodiments, provided herein is a method of cross-linking recombinant resilin, comprising: providing a composition comprising purified recombinant resilin; placing said recombinant resilin in a cross-linking solution comprising ammonium persulfate; and incubating said recombinant resilin in said cross-linking solution at a temperature of at least 60° C., thereby generating a cross-linked recombinant resilin solid composition.

In some embodiments, the incubation is performed for at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, or at least 2 hours.

In some embodiments, the recombinant resilin in said cross-linking solution is incubated at a temperature of from 60° C. to 85° C., from 70° C. to 85° C., or from 75° C. to 85° C.

In some embodiments, the incubation is performed for at least 2 hours.

In some embodiments, the cross-linking solution does not comprise a photocatalyst or a cross-linking enzyme.

In some embodiments, the cross-linked recombinant resilin solid composition is stable at room temperature for more than 5 days, more than 10 days, more than 20 days, or more than 40 days.

In some embodiments, the purified recombinant resilin is prepared by recombinantly expressing a gene encoding said recombinant resilin in a modified organism in a culture, and purifying expressed recombinant resilin from said culture.

Also provided herein, according to some embodiments, is a composition comprising cross-linked recombinant resilin, wherein said recombinant resilin has been cross-linked by exposure of said resilin to ammonium persulfate and heat. In some embodiments, the composition does not comprise a cross-linking enzyme. In some embodiments, the composition does not comprise a photocatalyst.

In some embodiments, provided herein is a recombinant resilin composition comprising a cross-linked recombinant resilin in a polar nonaqueous solvent.

In some embodiments, the polar nonaqueous solvent is a protic solvent. In some embodiments, the protic solvent is selected from the group consisting of: glycerol, propylene glycol, and ethylene glycol. In some embodiments, the polar nonaqueous solvent is an aprotic solvent. In some embodiments, the aprotic solvent is DMSO.

In some embodiments, the composition comprises from 20-40% resilin by weight.

In some embodiments, the resilin is at least 20%, at least 50%, at least 70%, or at least 80% full length resilin as a portion of all resilin as measured by size exclusion chromatography.

In some embodiments, the polar nonaqueous solvent is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% by volume of said solvent.

In some embodiments, the recombinant resilin composition has an elastic modulus greater than a similar cross-linked recombinant resilin in an aqueous solvent.

In some embodiments, the recombinant resilin composition comprises a hardness of at least 10 as measured using a Shore 00 Durometer via ASTM D2240.

In some embodiments, the recombinant resilin composition comprises a hardness of from about 10 to about 50 as measured using a Shore 00 Durometer via ASTM D2240.

In some embodiments, the recombinant resilin composition comprises a rebound resilience from about 40% to about 60% as measured by ASTM D7121.

In some embodiments, the recombinant resilin composition comprises a compressive stress at 25% of about 6 to about 8 psi as measured by ASTM D575.

In some embodiments, the recombinant resilin composition does not undergo an elastic to plastic transition below 2 kN of compressive force as measured by a Zwick compression test. In some embodiments, the recombinant resilin composition is a foam material.

Also provided herein, according to some embodiments, is a method of preparing a recombinant resilin solid, comprising: providing a cross-linked recombinant resilin solid composition in an aqueous solvent; and exchanging said aqueous solvent with a polar nonaqueous solvent.

In some embodiments, the polar nonaqueous solvent is a protic solvent. In some embodiments, the protic solvent is selected from the group consisting of: glycerol, propylene glycol, and ethylene glycol. In some embodiments, the polar nonaqueous solvent is an aprotic solvent. In some embodiments, the aprotic solvent is DMSO.

In some embodiments, the solvent exchange is performed for at least 8 hours, at least 16 hours, at least 24 hours, or at least 48 hours.

In some embodiments, the solvent exchange is performed at about 60° C.

In some embodiments, the cross-linked recombinant resilin solid composition comprises at least 20%, at least 50%, at least 70%, or at least 80% full length resilin as a portion of all resilin as measured by size exclusion chromatography.

In some embodiments, the cross-linked recombinant solid composition is prepared by the ammonium persulfate and heat method described herein.

Also provided herein, according to some embodiments, is a method of tuning material properties of a solid composition comprising cross-linked recombinant resilin solid, comprising: providing a cross-linked recombinant resilin solid composition comprising an aqueous solvent; and performing a solvent exchange to replace said aqueous solvent with a polar nonaqueous solvent. In some embodiments, replacing said solvent changes the elastic modulus, the resilience, the hardness, the maximum elastic compressive load, or the material lifetime of the cross-linked recombinant resilin solid composition.

Also provided herein, according to some embodiments, is a method of preparing a recombinant cross-linked resilin solid, comprising: providing a composition comprising purified recombinant resilin; placing said recombinant resilin in an aqueous solvent comprising ammonium persulfate; incubating said recombinant resilin in said aqueous solvent at a temperature of at least 60° C., thereby generating a cross-linked recombinant resilin solid composition; and exchanging said aqueous solvent with a polar nonaqueous solvent selected from the group consisting of: glycerol, propylene glycol, ethylene glycol, and DMSO.

Also provided herein, according to some embodiments, is a method of preparing a recombinant resilin foam, comprising: providing a cross-linked recombinant resilin solid composition in an aqueous solvent; exchanging said aqueous solvent with a polar nonaqueous solvent; and introducing one or more bubbles to the cross-linked recombinant resilin solid composition.

In some embodiments, the polar nonaqueous solvent is a protic solvent. In some embodiments, the protic solvent is selected from the group consisting of: glycerol, propylene glycol, and ethylene glycol. In some embodiments, the polar nonaqueous solvent is an aprotic solvent. In some embodiments, the aprotic solvent is DMSO.

In some embodiments, the solvent exchange is performed for at least 8 hours, at least 16 hours, at least 24 hours, or at least 48 hours.

In some embodiments, the solvent exchange is performed at about 60° C.

In some embodiments, the cross-linked recombinant resilin solid composition comprises at least 20%, at least 50%, at least 70%, or at least 80% full length resilin as a portion of all resilin as measured by size exclusion chromatography.

In some embodiments, the cross-linked recombinant solid composition is prepared by the ammonium persulfate and heat method described herein.

In some embodiments, the introducing comprises adding a blowing agent to the cross-linked recombinant resilin solid composition.

In some embodiments, the blowing agent comprises a chemical blowing agent. In some embodiments, the chemical blowing agent comprises sodium bicarbonate, potassium bicarbonate, ammonium, azodicarbonamide, isocyanate, hydrazine, isopropanol, 5-phenyltetrazole, triazole, 4,4′oxybis(benzenesulfonyl hydrazide) (OBSH), trihydrazine triazine (THT), hydrogen phosphate, tartaric acid, citric acid, and toluenesulphonyl semicarbazide (TSS). In some embodiments, the chemical blowing agent comprises sodium bicarbonate.

In some embodiments, the blowing agent comprises a physical blowing agent. In some embodiments, the physical blowing agent comprises chlorofluorocarbon (CFC), dissolved nitrogen, N₂, CH₄, H₂, CO₂, Ar, pentane, isopentane, hexane, methylene dichloride, and dichlorotetra-fluoroethane. In some embodiments, the physical blowing agent comprises dissolved nitrogen.

According to some embodiments, provided herein is a method for producing a composition comprising a recombinant resilin protein, the method comprising: culturing a population of recombinant host cells in a fermentation, wherein said recombinant host cells comprise a vector comprising a secreted resilin coding sequence, and wherein said recombinant host cells secrete a recombinant resilin protein encoded by said secreted resilin coding sequence; and purifying said recombinant resilin protein from said fermentation.

In some embodiments, the recombinant resilin protein is a full-length or truncated native resilin. In some embodiments, the native resilin is from an organism selected from the group consisting of: Drosophila sechellia, Acromyrmex echinatior, Aeshna, Haematobia irritans, Ctenocephalides fells, Bombus terrestris, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, Pediculus humanus corporis, Anopheles gambiae, Glossina morsitans, Atta cephalotes, Anopheles darlingi, Acyrthosiphon pisum, Drosophila virilis, Drosophila erecta, Lutzomyia longipalpis, Rhodnius prolixus, Solenopsis invicta, Culex quinquefasciatus, Bactrocera cucurbitae, and Trichogramma pretiosum. In some embodiments, the recombinant resilin protein comprises SEQ ID NO: 1. In some embodiments, the recombinant resilin protein comprises SEQ ID NO: 4.

In some embodiments, the recombinant resilin protein comprises an alpha mating factor secretion signal. In some embodiments, the recombinant resilin protein comprises a FLAG-tag. In some embodiments, the vector comprises more than one secreted resilin coding sequence.

In some embodiments, the recombinant host cells are yeast cells. In some embodiments, the yeast cells are methylotrophic yeast cells. In some embodiments, the recombinant host cells are a species selected from the group consisting of: Pichia (Komagataella) pastoris, Hansenula polymorpha, Arxula adeninivorans, Yarrowia lipolytica, Pichia (Scheffersomyces) stipitis, Pichia methanolica, Saccharomyces cerevisiae, and Kluyveromyces lactis.

In some embodiments, the recombinant host cells produce the recombinant resilin at a rate of greater than 2 mg resilin/g dry cell weight/hour. In some embodiments, the recombinant host cells produce a secreted fraction of the recombinant resilin that is greater than 50% as compared to the total recombinant resilin protein expressed by the recombinant host cells. In some embodiments, the recombinant host cells secrete the recombinant resilin at a rate of greater than 2 mg resilin/g dry cell weight/hour. In some embodiments, greater than 80% of the recombinant resilin is outside of the recombinant host cells in said fermentation. In some embodiments, the fermentation comprises at least 2 g recombinant resilin/L.

In some embodiments, purifying said recombinant resilin protein comprises: generating a first pellet fraction and a first supernatant fraction by centrifuging the fermentation; and isolating the recombinant resilin protein from the first pellet fraction. In some embodiments, purifying said recombinant resilin protein further comprises: adding a chaotrope to the first pellet fraction to generate a solution in which the recombinant resilin protein is soluble; generating a second supernatant fraction and a second pellet fraction by centrifuging the first pellet fraction comprising said chaotrope; and isolating the soluble full-length resilin from the second supernatant fraction.

In some embodiments, provided herein is a vector comprising a secreted resilin coding sequence. In some embodiments, the secreted resilin coding sequence encodes a full-length or truncated native resilin. In some embodiments, the secreted resilin coding sequence encodes a modified full-length or truncated native resilin. In some embodiments, the modified resilin comprises an addition, subtraction, replacement, or change in position of an amino acid residue capable of cross-linking to another resilin.

In some embodiments, the full-length or truncated native resilin is from an organism selected from the group consisting of: Drosophila sechellia, Acromyrmex echinatior, Aeshna, Haematobia irritans, Ctenocephalides fells, Bombus terrestris, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, Pediculus humanus corporis, Anopheles gambiae, Glossina morsitans, Atta cephalotes, Anopheles darlingi, Acyrthosiphon pisum, Drosophila virilis, Drosophila erecta, Lutzomyia longipalpis, Rhodnius prolixus, Solenopsis invicta, Culex quinquefasciatus, Bactrocera cucurbitae, and Trichogramma pretiosum.

In some embodiments, the secreted resilin coding sequences encodes a polypeptide comprising SEQ ID NO: 1. In some embodiments, the secreted resilin coding sequence encodes a polypeptide comprising SEQ ID NO: 4. In some embodiments, the secreted resilin coding sequence encodes a recombinant resilin comprising one or more A-repeats or quasi-A-repeats. In some embodiments, the secreted resilin coding sequence encodes a recombinant resilin comprising one or more B-repeats or quasi-B-repeats. In some embodiments, the secreted resilin coding sequence encodes a recombinant resilin comprising either one or more A-repeats or quasi-A-repeats or one or more B-repeats or quasi-B repeats but not both. In some embodiments, the secreted resilin coding sequence encodes a recombinant resilin comprising one or more A-repeats or quasi-A-repeats and one or more B-repeats or quasi-B-repeats.

In some embodiments, the recombinant resilin further comprises a chitin binding domain. In some embodiments, the secreted resilin coding sequence encodes a polypeptide comprising an alpha mating factor secretion signal. In some embodiments, the secreted resilin coding sequence comprises a FLAG-tag.

In some embodiments, the vector comprises more than one secreted resilin coding sequence. In some embodiments, the vector comprises 3 secreted resilin coding sequences. In some embodiments, the secreted resilin coding sequence is operatively linked to a constitutive or inducible promoter.

Also provided herein, according to some embodiments, is a recombinant host cell comprising one or more vectors comprising a secreted resilin coding sequence. In some embodiments, the recombinant host cell is a yeast cell. In some embodiments, the yeast cell is a methylotrophic yeast cell. In some embodiments, the recombinant host cell is a species selected from the group consisting of: Pichia (Komagataella) pastoris, Hansenula polymorpha, Arxula adeninivorans, Yarrowia lipolytica, Pichia (Scheffersomyces) stipitis, Pichia methanolica, Saccharomyces cerevisiae, and Kluyveromyces lactis.

In some embodiments, the recombinant host cell comprises 3 vectors comprising a secreted resilin coding sequence.

In some embodiments, the recombinant host cell produces recombinant resilin at a rate of greater than 2 mg resilin/g dry cell weight/hour. In some embodiments, the recombinant host cell has a secreted fraction of recombinant resilin that is greater than 50%. In some embodiments, the recombinant host cell secretes resilin at a rate of greater than 2 mg resilin/g dry cell weight/hour.

Also provided herein, according to some embodiments, is a fermentation comprising a recombinant host cell comprising one or more vectors comprising a secreted resilin coding sequence and a culture medium suitable for growing the recombinant host cell.

In some embodiments, the fermentation comprises at least 2 g recombinant resilin/L.

In some embodiments of the fermentation, greater than 80% of recombinant resilin is outside of the recombinant host cells.

In some embodiments of the fermentation, the recombinant resilin is full-length recombinant resilin.

Also provided herein, according to some embodiments, is a composition comprising recombinant resilin derived from a fermentation comprising a recombinant host cell comprising one or more vectors comprising a secreted resilin coding sequence and a culture medium suitable for growing the recombinant host cell. In some embodiments, the composition comprises at least 60% by weight of recombinant resilin.

In some embodiments, the composition has similar properties compared to compositions comprising similar amounts of native resilins. In some embodiments, the composition has different properties compared to compositions comprising similar amounts of native resilins.

In some embodiments, the composition comprises a resilience of greater than 50%. In some embodiments, the composition comprises has a compressive elastic modulus of less than 10 MPa. In some embodiments, the composition has a tensile elastic modulus of less than 10 MPa. In some embodiments, the composition has a shear modulus of less than 1 MPa. In some embodiments, the composition has an extension to break of greater than 1%. In some embodiments, the composition has a maximum tensile strength of greater than 0.1 kPa. In some embodiments, the composition has a Shore 00 Hardness of less than 90. In some embodiments, the composition comprises full-length resilin.

Also provided herein, according to some embodiments, is a method for producing a composition comprising recombinant resilin, the method comprising the step of culturing a recombinant host cell comprising one or more vectors comprising a secreted resilin coding sequence to produce a fermentation under conditions that promote secretion of recombinant resilin from the recombinant host cell.

In some embodiments, the method for producing a composition comprising recombinant resilin further comprises the step of purifying said recombinant resilin to produce full-length native resilin. In some embodiments, purifying said recombinant resilin to produce full-length native resilin comprises: generating a first pellet fraction and a first supernatant fraction by centrifuging the fermentation; and isolating the recombinant resilin protein from the first pellet fraction. In some embodiments, isolating the recombinant resilin protein from the first pellet fraction comprises: adding a chaotrope to the first pellet fraction to generate a solution in which the recombinant resilin protein is soluble; generating a second supernatant fraction and a second pellet fraction by centrifuging the first pellet fraction comprising said chaotrope; and isolating the recombinant resilin protein from the second supernatant fraction.

In some embodiments, the method for producing a composition comprising recombinant resilin further comprises the step of cross-linking a plurality of said recombinant resilins. In some embodiments, said cross-linking is enzymatic cross-linking. In some embodiments, said cross-linking is photochemical cross-linking. In some embodiments, the recombinant resilin protein comprises a full-length resilin protein.

Also provided herein, according to some embodiments, is a fermentation comprising a culture medium and a recombinant host cell, wherein the recombinant host cell comprises a vector, wherein the vector comprises a secreted resilin coding sequence, and wherein the recombinant host cell secretes recombinant resilin at a rate of at least 2 mg/g dry cell weight/hour.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead placed upon illustrating the principles of various embodiments of the invention.

FIG. 1 schematically illustrates the structure of an exemplary resilin.

FIG. 2 is a flow diagram of methods for the production of compositions comprising recombinant resilins.

FIG. 3 is an illustrative map of a vector that comprises 3 secreted resilin coding sequences.

FIG. 4 shows photographs of proteinaceous block co-polymers comprising cross-linked purified recombinant resilins in various shapes and forms.

FIG. 5 shows photographs of the compression of a proteinaceous block co-polymer comprising cross-linked recombinant resilin.

FIG. 6 shows the total degraded resilin as measured by ELISA using an antibody specific for the FLAG tag on resilin from resilin solid compositions cross-linked via ammonium persulfate plus heat (AP); ammonium persulfate plus heat and washed 3 times to remove excess cross-linking solution (AP 3× washed); and enzymatic cross-linking via horseradish peroxidase (HRP).

FIG. 7 shows a photograph of resilin foam with 10 wt % fumed silica (left) and 5 wt % fumed silica (right).

FIG. 8 shows a photograph of resilin foam with 5 wt % fumed silica. A quarter dollar is included for comparison.

FIG. 9 shows an image of glycerol-based and water-based cross-linked resilin compositions after cross-linking (top) and after 7 days of unsealed storage for one week (bottom).

FIGS. 10A, 10B, and 10C depict stress strain curves as measured by a rheometer for the following cross-linked resilin compositions: 27% by weight resilin in a glycerol solvent prepared by a 60% glycerol solvent exchange followed by a 100% glycerol solvent exchange (FIG. 10A); 27% by weight resilin in a propylene glycol solvent (FIG. 10B); and 27% full length resilin (i.e., about 80% full length resilin as a proportion of all resilin) in a propylene glycol solvent (FIG. 10C).

FIG. 11 shows a stress-strain curve of resilin foam with 5 wt % fumed silica.

FIG. 12 shows a plot of the relative values of resilience and elastic modulus as measured by a rheometer for each of the following cross-linked resilin solid compositions: 20 wt % resilin, propylene glycol (“A”); 27 wt % resilin, propylene glycol (“B”); 40 wt % resilin, propylene glycol (“C”); resilin coacervate, propylene glycol (“D”); 27 wt % full-length resilin, propylene glycol (“E”); 27 wt % resilin, 60-100% glycerol (“F”); 27 wt % resilin, 100% glycerol (“H”); and 27 wt % resilin, ethylene glycol(“I”).

FIG. 13 shows a plot of applied force vs. strain curves as measured from the Zwick tensile tester for each of the following cross-linked resilin solids: 27 wt % resilin ethylene glycol solid (“A”), 27 wt % resilin propylene glycol solid (“C), coacervate resilin propylene glycol solid (“D”), 27 wt % resilin 60-100% glycerol solid (“G”), and 27 wt % resilin 100% glycerol solid (“_(H)”).

The figures depict various embodiments of the present disclosure for purposes of illustration only. One skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles described herein.

DETAILED DESCRIPTION

The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure pertains.

The terms “a” and “an” and “the” and similar referents as used herein refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The term “about,” “approximately,” or “similar to” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, or on the limitations of the measurement system. It should be understood that all ranges and quantities described below are approximations and are not intended to limit the invention. Where ranges and numbers are used these can be approximate to include statistical ranges or measurement errors or variation. In some embodiments, for instance, measurements could be plus or minus 10%.

Amino acids can be referred to by their single-letter codes or by their three-letter codes. The single-letter codes, amino acid names, and three-letter codes are as follows: G—Glycine (Gly), P—Proline (Pro), A—Alanine (Ala), V—Valine (Val), L—Leucine (Leu), I—Isoleucine (Ile), M—Methionine (Met), C—Cysteine (Cys), F—Phenylalanine (Phe), Y—Tyrosine (Tyr), W—Tryptophan (Trp), H—Histidine (His), K—Lysine (Lys), R—Arginine (Arg), Q—Glutamine (Gln), N—Asparagine (Asn), E—Glutamic Acid (Glu), D—Aspartic Acid (Asp), S—Serine (Ser), T—Threonine (Thr).

The terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are intended to be inclusive in a manner similar to the term “comprising.”

The term “microbe” as used herein refers to a microorganism, and refers to a unicellular organism. As used herein, the term includes all bacteria, all archaea, unicellular protista, unicellular animals, unicellular plants, unicellular fungi, unicellular algae, all protozoa, and all chromista.

The term “native” as used herein refers to compositions found in nature in their natural, unmodified state.

The terms “optional” or “optionally” mean that the feature or structure may or may not be present, or that an event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where the event or circumstance does not occur.

The term “secreted fraction” as used herein refers to the fraction of recombinant resilins that are secreted from cells compared to the total resilins produced by the cells.

The term “secretion signal” as used herein refers to a short peptide that when fused to a polypeptide mediates the secretion of that polypeptide from a cell.

The term “secreted resilin coding sequence” as used herein refers to a nucleotide sequence that encodes a resilin as provided herein fused at its N-terminus to a secretion signal and optionally at its C-terminus to a tag peptide or polypeptide.

The term “recombinant” as used herein in reference to a polypeptide (e.g., resilin) refers to a polypeptide that is produced in a recombinant host cell, or to a polypeptide that is synthesized from a recombinant nucleic acid.

The term “recombinant host cell” as used herein refers to a host cell that comprises a recombinant nucleic acid.

The term “recombinant nucleic acid” as used herein refers to a nucleic acid that is removed from its naturally occurring environment, or a nucleic acid that is not associated with all or a portion of a nucleic acid abutting or proximal to the nucleic acid when it is found in nature, or a nucleic acid that is operatively linked to a nucleic acid that it is not linked to in nature, or a nucleic acid that does not occur in nature, or a nucleic acid that contains a modification that is not found in that nucleic acid in nature (e.g., insertion, deletion, or point mutation introduced artificially, e.g., by human intervention), or a nucleic acid that is integrated into a chromosome at a heterologous site. The term includes cloned DNA isolates and nucleic acids that comprise chemically-synthesized nucleotide analog.

The term “vector” as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which generally refers to a circular double stranded DNA loop into which additional DNA segments can be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. Other vectors include bacteriophages, cosmids, bacterial artificial chromosomes (BAC), and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a cell into which they are introduced (e.g., vectors having an origin of replication that functions in the cell). Other vectors can be integrated into the genome of a cell upon introduction into the cell, and are thereby replicated along with the cell genome.

The term “repeat” as used herein, in reference to an amino acid or nucleic acid sequence, refers to a sub-sequence that is present more than once in a polynucleotide or polypeptide (e.g., a concatenated sequence). A polynucleotide or polypeptide can have a direct repetition of the repeat sequence without any intervening sequence, or can have non-consecutive repetition of the repeat sequence with intervening sequences. The term “quasi-repeat” as used herein, in reference to amino acid or nucleic acid sequences, is a sub-sequence that is inexactly repeated (i.e., wherein some portion of the quasi-repeat subsequence is variable between quasi-repeats) across a polynucleotide or polypeptide. Repeating polypeptides and DNA molecules (or portions of polypeptides or DNA molecules) can be made up of either repeat sub-sequences (i.e., exact repeats) or quasi-repeat sub-sequences (i.e., inexact repeats).

The term “native resilin” as used herein refers to an elastomeric polypeptide or protein produced by insects. GenBank Accession Nos. of non-limiting examples of native resilin includes the following NCBI sequence numbers: NP 995860 (Drosophila melanogaster), NP 611157 (Drosophila melanogaster), Q9V7U0 (Drosophila melanogaster), AAS64829, AAF57953 (Drosophila melanogaster), XP 001817028 (Tribolium castaneum) and XP001947408 (Acyrthosiphon pisum).

The term “modified” as used herein refers to a protein or polypeptide sequence that differs in composition from a native protein or polypeptide sequence, where the functional properties are preserved to within 10% of the native protein or polypeptide properties. In some embodiments, the difference between the modified protein or polypeptide and the native protein or polypeptide can be in primary sequence (e.g., one or more amino acids are removed, inserted or substituted) or post-translation modifications (e.g., glycosylation, phosphorylation). Amino acid deletion refers to removal of one or more amino acids from a protein. Amino acid insertion refers to one or more amino acid residues being introduced in a protein or polypeptide. Amino acid insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Amino acid substitution includes non-conservative or conservative substitution, where conservative amino acid substitution tables are well known in the art (see for example Creighton (1984) Proteins. W. H. Freeman and Company (Eds)). In some embodiments, the modified protein or polypeptide and the native protein or polypeptide amino acid or nucleotide sequence identity is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the amino acids or nucleotide bases.

The term “truncated” as used herein refers to a protein or polypeptide sequence that is shorter in length than a native protein or polypeptide. In some embodiments, the truncated protein or polypeptide can be greater than 10%, or greater than 20%, or greater than 30%, or greater than 40%, or greater than 50%, or greater than 60%, or greater than 70%, or greater than 80%, or greater than 90% of the length of the native protein or polypeptide.

The term “homolog” or “substantial similarity,” as used herein, when referring to a polypeptide, nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate amino acid or nucleotide insertions or deletions with another amino acid or nucleic acid (or its complementary strand), there is amino acid or nucleotide sequence identity in at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the amino acids or nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

The term “resilin” as used herein refers to a protein or a polypeptide, capable of cross-linking to form an elastomer, where the protein or polypeptide is a native resilin, or a native resilin that is modified, or a native resilin that is truncated. Resilins of the present invention are preferably recombinant resilins. In some embodiments, recombinant resilins comprise a natural or modified (e.g., truncated or concatenated) nucleotide sequence coding for resilin or resilin fragments (e.g., isolated from insects), heterologously expressed and secreted from a host cell. In preferred embodiments, the secreted recombinant resilin protein is collected from a solution extracellular to the host cell. In some embodiments, resilin is in a form of a foam material, e.g., a solid foam.

As used herein, the term “elastomer” refers to a polymer with viscoelasticity and typically weak inter-molecular forces (except for covalent cross-links between molecules, if they are present). Viscoelasticity is a property of materials that exhibit both viscous and elastic characteristics when undergoing deformation, and therefore exhibit time-dependent strain. Elasticity is associated with bond stretching along crystallographic planes in an ordered solid, and viscosity is the result of the diffusion of atoms or molecules inside an amorphous material. Elastomers that are viscoelastic, therefore, generally have low Young's modulus and high failure strain compared with other materials. Due to the viscous component of the material, viscoelastic materials dissipate energy when a load is applied and then removed. This phenomenon is observed as hysteresis in the stress-strain curve of viscoelastic materials. As a load is applied there is a particular stress-strain curve, and as the load is removed the stress-strain curve upon unloading is different than that of the curve during loading. The energy dissipated is the area between the loading and unloading curves.

As used herein, the term “nonaqueous” refers to a solvent that predominantly comprises one or more compounds that are not water. This includes compositions that have undergone a solvent exchange process with a solvent that results in an overall decrease in the proportion of water present as a solvent, i.e., water has been replaced by non-water molecules as a solvent. In some embodiments, a nonaqueous solvent is one that comprises less than 50% water. A polar nonaqueous solvent, as used herein with respect to solvents for cross-linked resilin compositions, refers to any nonaqueous solvent that is capable of dissolving resilin.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value inclusively falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

Resilin Compositions

Provided herein are compositions comprising recombinant resilins, and methods for their production.

Resilins have many unique properties compared to petroleum-based elastomers. Most notably, resilin has an extreme elastic efficiency (i.e., resilience), where very little of the energy input into deformation is lost as heat. Other desirable properties of resilin include, for example, compressive elastic modulus, tensile elastic modulus, shear modulus, hardness, rebound, and compression set. Moreover, resilin is a protein, and therefore can be biodegraded, which makes it more environmentally friendly than petroleum-based polymers. Also, resilin is biocompatible and can therefore be used in applications that involve contact with humans or animals. Lastly, the mechanical properties of recombinant resilins can be tuned through varying protein sequence, protein structure, amount of intermolecular cross-linking and processing variables to produce elastomers designed for a universe of specific applications.

In some embodiments, provided herein are cross-linked resilin compositions with desirable mechanical properties and methods of producing them. In some embodiments, provided herein are methods of cross-linking resilin compositions to form a cross-linked resilin solid that can be performed efficiently in large batches and is not susceptible to degradation from impurities left over from the cross-linking reaction. In some embodiments, the cross-linking reaction comprises exposure of the resilin to a persulfate, such as ammonium persulfate. Heat can be applied to initiate a cross-linking reaction catalyzed by persulfate. This type of cross-linking reaction does not leave any photoactive or enzymatic compounds in the composition. Furthermore, this cross-linking reaction does not require photoactivation, so large batches can be produced efficiently without the requirement for light to reach all parts of the cross-linking solution. In some embodiments, cross-linking occurs in vessels or molds such that the recombinant resilin compositions obtained have specific shapes or forms.

The cross-linked resilin solid compositions provided herein also include cross-linked resilin compositions comprising a polar nonaqueous solvent to provide desired mechanical properties, such as elastic modulus, hardness, maximum elastic compressive load, resilience, and material lifetime/fatigue that are preferred in certain applications. In some embodiments, the compositions are made by performing a solvent exchange with a resilin composition to replace an aqueous solvent with a nonaqueous solvent. Solvents that are capable of doing solvent exchange with cross-linked resilin include solvents that dissolve resilin in its non-crosslinked form.

In preferred embodiments, the nonaqueous solvent is non-volatile and water soluble or polar. In some embodiments, the molecular weight of the solvent is about 100 or less. In some embodiments, the polar nonaqueous solvent comprises glycerol, propylene glycol, ethylene glycol, or DMSO. In some embodiments, the solvent exchange is done using a polar nonaqueous solvent gradient (e.g., from 60% to 100% glycerol).

In some embodiments, the cross-linked resilin compositions described herein can be used to provide a composition having improved physical properties, including, e.g., for absorption of energy from an applied force as desired. In some embodiments, the cross-linked resilin compositions described herein can be used to improve existing products containing rubber. In particular, some of the cross-linked resilin compositions provided herein can absorb a large amount of force, while not transitioning to an inelastic material.

In some embodiments, the cross-linked resilin compositions provided herein can be used as a mid-sole. In some embodiments, the cross-linked resilin compositions provided herein can be used as part of a core for a golf ball. In other embodiments, the cross-linked resilin compositions provided herein can be used in handles or grips, e.g., for sports equipment such as golf clubs or tennis rackets, as bicycle grips or motorcycle grips, or as groups for tools and industrial uses such as hammers, nail guns, jackhammers, and any other tools where it is preferable to absorb and return energy. In some embodiments, the cross-linked resilin compositions provided herein can be used in brushings or dampenings, e.g., skate board trucks or hard drive platter vibration dampener. In some embodiments, the cross-linked resilin compositions can be used as superior material for wheels, such as for skate boards, roller blades, or scooters. In some embodiments, the cross-linked resilin compositions provided herein can be used for safety and protective gear, such as padding for protective equipment such as helmets, elbow or knee pads, or hard hats, or as a protective outer layer to protect the skin from abrasions.

In some embodiments, the cross-linked resilin compositions provided herein can be used for automotive parts, e.g., suspension components such as bushings or shock absorbers, or for interior cushioning such as seat bolsters and lumbar support. In some embodiments, the cross-linked resilin compositions provided herein can be used for tires and inner tubes. In some embodiments, the cross-linked resilin compositions provided herein can be used for suberballs. In some embodiments, the cross-linked resilin compositions provided herein can be used for shoe insoles, midsoles, and outsoles. In some embodiments, the cross-linked resilin compositions provided herein can be used in a padded mat. In some embodiments, the cross-linked resilin compositions provided herein can be used for several types of gaskets or O-rings. In some embodiments, the cross-linked resilin compositions provided herein can be added to plastic items to increase their impact resistance. In some embodiments, the cross-linked resilin compositions provided herein can be used for protective cases, such as phone or tablet cases. In some embodiments, the cross-linked resilin compositions provided herein can be used for rubber stamps. In some embodiments, the cross-linked resilin compositions provided herein can be used for rollers. In some embodiments, the cross-linked resilin compositions provided herein can be used for rubber bands.

In some embodiments, the cross-linked resilin compositions provided herein can be used for shoe soles, basement flooring, noise protection for sound studios, car bumpers, cushion pads, door mats, yoga mats, drum pads, window wipers, car tires, fire hoses, electrical wiring insulation, rubber bands, rubber ducks, elastic gloves, cooking utensils, rain boots, teething toys, bicycle tires, watches, jars, gaskets, hair ties, flip-flops, phone cases, medicine balls, bouncy balls, seals for electronic devices to prevent contamination from water or dust, refrigerator or freezer door seals, seals to prevent air flow in or out of a chamber, trampolines, pacifiers, window seals, halloween masks, garden hoses, table tennis rackets, conveyer belts, ducting, stamps, balloons, cosmetic compositions for the care and protection of skin and hair, nail compositions, or preparations for decorative cosmetics.

Suitable skin cosmetic compositions are, for example, face tonics, facial masks, e.g., sheet masks, deodorants, and other cosmetic lotions. Compositions for use in decorative cosmetics include, for example, concealing sticks, stage make-up, mascara and eye shadows, lipsticks, kohl pencils, eyeliners, blushers, powders, and eyebrow pencils.

Depending on the field of use, the compositions described herein can be applied in a form suitable for skincare, such as, for example, as cream, foam, gel, stick, mousse, milk, spray (pump spray or propellant-containing spray) or lotion.

FIG. 1 illustrates an example of a native resilin, which contains an N-terminal A-domain comprising a plurality of repeat units comprising the consensus amino acid sequence YGXP (“A-repeat”), where X is any amino acid; a chitin-binding type RR-2 (C) domain (Pfam reference PF00379; Rebers JE & Willis, JI-1. A conserved domain in anthropod cuticular proteins binds chitin. Insect Biochem Mol Biol 31:1083-1093); and a C-terminal B-domain comprising a plurality of repeat units comprising the consensus amino acid sequence UYZXZ (“B-repeat”), where U is glycine or serine; Z is serine, glycine, arginine, or proline; and X is any amino acid. Not all naturally occurring resilins have A-, C-, and B-domains. Native resilins produced by various insects typically have inexact repeats (i.e., quasi-repeats) within the A- and/or B-domains with some amino acid variation between the quasi-repeats.

In some embodiments, the recombinant resilins provided herein comprise one or more A-repeats. In some embodiments, the recombinant resilins comprise N-terminal A-domains comprising a plurality of blocks of A-repeat and/or quasi-A-repeat amino acid sub-sequences each with the consensus sequence SXXYGXP, where S is serine, X is an amino acid, Y is tyrosine, G is glycine, and P is proline.

In some embodiments, the recombinant resilins provided herein comprise one or more B-repeats. In some embodiments, the recombinant resilins comprise a C-terminal B-domain comprising a plurality of blocks of B-repeat and/or quasi-B-repeat amino acid sub-sequences each with the consensus sequence GYZXZZX and/or SYZXZZX, where G is glycine; Y is tyrosine; Z is serine, glycine, proline, or arginine; S is serine; and X is any amino acid.

In some embodiments, the recombinant resilins provided herein comprise one or more A-repeats. In some such embodiments, the recombinant resilins comprise between 1 and 100 A-repeats, or from 2 to 50 A-repeats, or from 5 to 50 A-repeats, or from 5 to 20 A-repeats.

In some embodiments, the recombinant resilins comprise one or more consensus sequences described by the formula,

(X₁-X₂-X₃-X₄)_(n)  (1)

wherein the brackets delineate a repeat or quasi-repeat of the consensus sequence;

wherein n describes the number of A-repeats or quasi-A-repeats, and is from 1 to 100, or from 2 to 50, or from 5 to 50, or from 5 to 20;

wherein X₁ is a motif that is 4 amino acids in length, wherein the first amino acid of X₁ is Y, and wherein the remaining amino acids of X₁ are GAP, GLP, GPP, GTP, or GVP;

wherein X₂ is a motif that is from 3 to 20 amino acids in length;

wherein X₂ comprises GGG, GGGG, N, NG, NN, NGN, NGNG, GQGG, GQGN, GQGQ, GQGQG, or 3 or more glycine residues, or wherein 50% or more of the residues of X₂ are either glycine or asparagine, or wherein 60% or more of the residues of X₂ are either glycine or asparagine, or wherein 70% or more of the residues of X₂ are either glycine or asparagine, or wherein 80% or more of the residues of X₂ are either glycine or asparagine;

wherein X₃ is a motif that is from 2 to 6 amino acids in length, wherein X₃ is GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG; and

wherein X₄ is a motif that is from 1 to 2 amino acids in length, wherein X₄ is S, D, T, N, L, DS, DT, LS, SS, ST, TN, or TS.

In some such embodiments, the recombinant resilins comprise motifs X₁, X₂, X₃, and X₄ whereas in other embodiments, the recombinant resilins comprise motifs X₁, X₂, X₃, or X₄, or combinations thereof.

In some embodiments, the recombinant resilins provided herein comprise one or more B-repeats. In some such embodiments, the recombinant resilins comprise between 1 and 100 B-repeats, or from 2 to 50 A-repeats, or from 5 to 50 A-repeats, or from 5 to 20 A-repeats.

In some embodiments, the recombinant resilins comprise one or more consensus sequences described by the formula,

(X₁₁-X₁₂-X₁₃)_(m)  (2)

wherein the brackets delineate a repeat or quasi-repeat of the consensus sequence;

wherein m describes the number of B-repeats or quasi-B-repeats, and is from 1 to 100;

wherein X₁₁ is a motif that is from 1 to 5 amino acids in length, the first amino acid is Y, and where the remaining amino acids can comprise GAP, GPP, SSG, or SGG;

wherein X₁₂ is a motif that is from 2 to 5 amino acids in length and comprises GQ, GN, RPG, RPGGQ, RPGGN, SSS, SKG, or SN; and

wherein X₁₃ is a motif that is from 4 to 30 amino acids in length and comprises GG, DLG, GFG, GGG, RDG, SGG, SSS, GGSF, GNGG, GGAGG, or 3 or more glycine residues, or 30% or more of the residues are glycine, or 40% or more of the residues are glycine, or 50% or more of the residues are glycine, or 60% or more of the residues are glycine.

In some such embodiments, the recombinant resilins comprise motifs X₁₁, X₁₂, and X₁₃ whereas in other such embodiments, the recombinant resilins comprise motifs X₁₁, X₁₂, or X₁₃, or combinations thereof.

In some embodiments, the recombinant resilins provided herein comprise one or more A-repeats, one or more B-repeats, and/or one or more C-domain. In some embodiments, the recombinant resilins comprise one or more A-repeats or one or more B-repeats but not both. In some embodiments, the recombinant resilins comprise one or more A-repeats but not B-repeats or C-domains. In some embodiments, the recombinant resilins comprise one or more B-repeats but not A-repeats or C-domains. In embodiments in which the recombinant resilins comprise a C-domain, the C-domain can be situated either on the N-terminal or the C-terminal sides of the A-repeats or B-repeats, or between the A-repeats and the B-repeats.

In some embodiments, the recombinant resilins further comprise the sequence XXEPPVSYLPPS, where X is any amino acid. In some such embodiments, the sequence is located on the N-terminal side of an A-repeat or B-repeat.

In some embodiments, the recombinant resilins are full-length native resilins expressed in a non-native environment. In some embodiments, the recombinant resilins comprise a truncated version of native resilins. In some embodiments, the truncated native resilins comprise at least one A-repeat. In some embodiments, the truncated native resilins comprise at least one B-repeat. Non-limiting examples of full-length and truncated native resilins are provided as SEQ ID NOs: 1 through 44. In some embodiments, the recombinant resilins are full-length Drosophila sechellia resilin (SEQ ID NO: 1). In some embodiments, the recombinant resilins are truncated Acromyrmex echinatior resilin (SEQ ID NO: 4). In some embodiments, the recombinant resilins are full-length or truncated native resilins that are cross-linked in a non-native manner (e.g., less or more cross-linking, cross-linking via different amino acid residues).

In some embodiments, the recombinant resilins are modified full-length or truncated native resilins. In some embodiments, the recombinant resilins are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to a full-length or truncated native resilin. In some embodiments, the recombinant resilins are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to full-length Drosophila sechellia resilin (SEQ ID NO: 1). In some embodiments, the recombinant resilins are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to truncated Acromyrmex echinatior resilin (SEQ ID NO: 4).

There are a number of different algorithms known in the art which can be used to measure nucleotide sequence or protein sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. See, e.g., Pearson, Methods Enzymol. 183:63-98, 1990 (hereby incorporated by reference in its entirety). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference. Alternatively, sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410, 1990; Gish and States, Nature Genet. 3:266-272, 1993; Madden et al., Meth. Enzymol. 266:131-141, 1996; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Zhang and Madden, Genome Res. 7:649-656, 1997, especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997.

In some embodiments, the modified resilins differ from full-length or truncated native resilins in amino acid residues that are post-translationally modified (e.g., glycosylated, phosphorylated) such that the modified resilins have one or more different locations and/or different amounts and/or different types of post-translational modifications than the full-length or truncated native resilins. In some embodiments, the modified resilins differ from full-length or truncated native resilins in amino acid residues that are involved in cross-linking such that the modified resilins have one or more different locations and/or different amounts and/or different types of amino acids that are involved in cross-linking than full-length or truncated native resilins. In some such embodiments, the modified resilins differ from the full-length or truncated native resilin in comprising one or more additional or fewer tyrosine residues, one or more additional or fewer lysine residues, and/or one or more additional or fewer cysteine residues.

In some embodiments, the recombinant resilins comprise concatenated native or truncated native resilins or concatenated modified resilins. In some embodiments, the concatenated native or truncated native resilins or concatenated modified resilins comprise at least 2 A-repeats (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the concatenated truncated native resilins or concatenated modified resilins comprise at least 2 B-repeats (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more).

Protein Quantification and Purity Analysis

In some embodiments, the total amount of full length resilin in a composition is determined by size exclusion chromatography (SEC), Reverse Phase High Performance Liquid Chromatography or other methods as known in the art (e.g. quantitative Western blots). In various embodiments, the same or similar assays may be used to assess the relative amount of full length resilin in its monomeric and aggregate forms.

As described herein, in a specific embodiment, data characterizing the relative amount of the predominant species of resilin (i.e. monomeric full length resilin) in a resilin composition is determined from Size Exclusion Chromatography (SEC) as follows: resilin powder is dissolved in 5M Guanidine Thiocyanate and injected onto a Yarra SEC-3000 SEC-HPLC column to separate constituents on the basis of molecular weight. Refractive index is used as the detection modality. BSA is used as a general protein standard with the assumption that >90% of all proteins demonstrate do/dc values (the response factor of refractive index) within ˜7% of each other. Poly(ethylene oxide) is used as a retention time standard, and a BSA calibrator is used as a check standard to ensure consistent performance of the method. A range corresponding to monomeric full length resilin is assessed from 60-40 kDa; higher peaks observed which may correspond to aggregate or polymerized full length resilin are not included in this quantification. The relative percentage of this resilin peak is reported as mass % and area % to determine the amount of full length resilin in a composition as a portion of total resilin.

Cross-Linking

In some embodiments, the methods provided herein further comprise the step of cross-linking the recombinant resilins to obtain the recombinant resilin compositions provided herein (step 1005 in FIG. 2). The recombinant resilin in the desired solvent with cross-linking agents can be filled into small shaped molds to control the shape of the resulting solid after cross-linking. Examples of resulting recombinant resilin solids are shown in FIG. 4.

In some embodiments, cross-linking is achieved via tyrosine residues. Resilin has a tyrosine residue every 15-24 amino acids throughout most of its length. In some embodiments, cross-linking of resilin creates di- and tri-tyrosine crosslinking in resilin to form a resilin solid.

In other embodiments, cross linking is achieved via lysine residues. In some embodiments, cross linking is achieved via cysteine residues. In some embodiments, cross-linking employs transglutaminase (see, for example, Kim Y, Gill E E, Liu J C. Enzymatic Cross-Linking of Resilin-Based Proteins for Vascular Tissue Engineering Applications. Biomacromolecules. 17(8):2530-9). In some embodiments, cross-linking employs poly(ethylene glycol) (PEG) (McGann C L, Levenson E A, Kiick K L. Macromol. Chem. Phys. 2013, 214, 203-13; McGann C L, Akins R E, Kiick K L. Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure. Biomacromolecules. 2016; 17(1):128-40).

In some embodiments, recombinant resilin is cross-linked via enzymatic cross-linking (e.g., using horseradish peroxidase). While this method can efficiently cross-link large solutions of resilin, the resulting cross-linked product comprises covalently incorporated active enzyme in the cross-linked resilin solid. This yields radical chain reactions that degrade the protein backbone of the resilin.

In other embodiments, recombinant resilin is cross-linked via photochemical cross-linking (see, for example, Elvin C M, Carr A G, Huson M G, Maxwell J M, Pearson R D, Vuocolo T, Liyou N E, Wong D C C, Merritt D J, Dixon N E. Nature 2005, 437, 999-1002; Whittaker J L, Duna N K, Elvin C M, Choudhury N R. Journal of Materials Chemistry B 2015, 3, 6576-79; Degtyar E, Mlynarczyk B, Fratzl P, Harrington M J. Polymer 2015, 69, 255-63).

This cross-linking reaction also results in the incorporation of an active catalyst into the resilin solid that is difficult to completely dialyze out of the material, uses relatively high catalyst loading, and is not efficient for reactions where photoactivation throughout the mold may be difficult.

Provided herein are new recombinant resilin cross-linking chemistries that prevent degradation and make solid substances with some mechanical properties preferred for certain applications where the amount and form of energy absorption is important.

In preferred embodiments, recombinant resilin is cross-linked via a solvent comprising ammonium persulfate and application of heat (e.g., incubation at a temperature of about 80° C. for about 2.5 hours). This cross-linking reaction is efficient and does not leave an active catalyst behind in the resilin solid, resulting in a composition with less degraded protein.

In some embodiments, the final concentration of ammonium persulfate for the resilin cross-linking reaction is about 30 to 40 mM. In some embodiments, the cross-linking solution with ammonium persulfate and resilin is incubated (i.e., exposed to heat) for at least 15 minutes, at least 30 minutes, at least 60 minutes, at least 90 minutes, or at least 2 hours to facilitate sufficient cross-linking to form a solid with desirable mechanical properties. In some embodiments, other persulfates are used.

In some embodiments, the heat applied to the ammonium persulfate and resilin composition solution is at least 40° C., at least 45° C., at least 50° C., at least 55° C., at least 60° C., at least 65° C., at least 70° C., at least 75° C., or at least 80° C. In some embodiments, the heat applied to the cross-linking solution of ammonium persulfate and resilin is from 50° C.-90° C. or from 60° C.-90° C. or from 70° C.-90° C. or from 75° C. to 85° C. or from 70° C. to 80° C. In some embodiments, the cross-linking solution does not comprise an enzymatic catalyst. In some embodiments, the cross-linking solution does not comprise a photoactive catalyst.

In some embodiments, the cross-linking solution comprises at least 1%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% resilin, from 1% to 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%; from 10% to 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%; from 20% to 100%, 90%, 80%, 70%, 60%, 50%, 40%, or 30%; from 30% to 100%, 90%, 80%, 70%, 60%, 50%, or 40%; from 40% to 100%, 90%, 80%, 70%, 60%, or 50%; from 50% to 100%, 90%, 80%, 70%, or 60%; from 60% to 100%, 90%, 80%, or 70%; from 70% to 100%, 90%, or 80%; from 80% to 100%, or 90%; or from 90% to 100% by weight of the total cross-linking solution. In preferred embodiments, resilin is dissolved in a cross-linking solution to a concentration of between 20% to 30% by weight resilin. In some embodiments the total resilin comprises at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% full length resilin as measured by size exclusion chromatography (SEC).

Solvent-Exchanged Resilin Solids

Cross-linked resilin can be formed in an aqueous solvent resulting in a composition that has a low hardness and elastic modulus that is less suitable for certain applications where energy absorption and stiffness are desired. In some embodiments, a solvent exchange is performed on cross-linked resilin compositions to replace an aqueous solvent with a polar nonaqueous solvent to provide desired material properties. In some embodiments, the polar nonaqueous solvent comprises glycerol, propylene glycol, ethylene glycol, or DMSO. In some embodiments, the nonaqueous solvents are water-miscible. In some embodiments the nonaqueous solvents are non-volatile solvents. In some embodiments, the nonaqueous solvents comprise molecules with a plurality of alcohol functional groups. In some embodiments the nonaqueous solvent comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of a molecule or molecules with a molecular weight less than about 100 g/mol. As described herein, material properties of cross-linked resilin compositions, including elastic modulus, hardness, maximum elastic compressive load, resilience, and material lifetime/fatigue, can be tuned using solvent exchange. Solvents that are capable of doing solvent exchange with cross-linked resilin include solvents that dissolve resilin in its non-crosslinked form.

In some embodiments, a solvent exchange to replace an aqueous solvent of resilin with a polar nonaqueous solvent is performed in the presence of heat, e.g., at a temperature of about 60° C. In some embodiments the solvent exchange process is performed in a solution containing at least 1×, at least 2×, at least 5×, at least 10×, or at least 20× the volume of exchange solvent relative to the resilin solid. In some embodiments, the solvent exchange is performed for at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 24 hours, or at least 48 hours. In some embodiments, glycerol, propylene glycol, ethylene glycol, or DMSO are used as exchange solvents for cross-linked resilin solid compositions.

In some embodiments, the choice of exchange solvent and the concentration used is selected to achieve a desired tunable mechanical property, such as stiffness, from the solvent design. This can be selected depending on the desired application (e.g., shoe soles, golf balls, etc.).

In some embodiments, the cross-linked resilin composition formed by the above solvent exchange process and in a nonaqueous solvent is stable at room temperature for at least 10 days, at least 20 days, at least 30 days, at least 40 days, or at least 50 days. In some embodiments, the cross-linked resilin composition formed by the above solvent exchange process and in a nonaqueous solvent is resistant to mold growth for at least 10 days, at least 20 days, at least 30 days, at least 40 days, or at least 50 days. In some embodiments, the cross-linked resilin composition formed by the above solvent exchange process and in a nonaqueous solvent is resistant to dehydration for at least 7 days or at least 14 days. By contrast, water-based cross-linked resilin solids were observed to dehydrate completely within 2 weeks and to grow mold within 5-14 days when stored in an open environment under ambient conditions. In addition, a 10 mm by 5 mm disc of water-based resilin solid breaks when 220 lbs of force is applied whereas a 10 mm by 5 mm disc of a glycerol-based resilin solid does not break under 220 lbs of force.

In some embodiments, relative material properties between consecutively tested materials can be tested using a rheometer. For example, a cross-linked resilin composition may be formed in a cylinder mold, and the resulting resilin cylinder is subjected to compression test using a rheometer. A recombinant resilin cylinder could be compressed from an initial height of 7.3 mm (avg width 5.4 mm) to less than 0.66 mm without any breakage. As shown in FIG. 5, the cylinder returned to a height of 6.7 mm (avg width 5.6 mm) upon release of the compressive load.

In some embodiments, the polar nonaqueous solvent comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of a polar nonaqueous composition by volume of said solvent. In some embodiments, the solvent comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of glycerol, propylene glycol, ethylene glycol, or DMSO by volume of said solvent.

In some embodiments, the recombinant resilin composition provided herein has an elastic modulus greater than cross-linked recombinant resilin in an aqueous solvent.

In some embodiments, the cross-linked resilin compositions provided herein have a Shore 00 Hardness of 50 or more, 40 or more, 30 or more, 20 or more, 10 or more, from 10 to 50, 40, 30, or 20; from 20 to 50, 40, or 30; from 30 to 50 or 40; or from 40 to 50; In some embodiments, hardness measurements in resilin are performed according to ASTM D2240. In some embodiments, the recombinant resilin composition comprises a hardness of at least 10 as measured using a Shore OO Durometer via ASTM D2240. In some embodiments, the recombinant resilin composition comprises a hardness of at least 30 as measured using a Shore OO Durometer via ASTM D2240. In some embodiments, the recombinant resilin composition comprises a hardness of from about 10 to about 50 as measured using a Shore OO Durometer via ASTM D2240. In some embodiments, the hardness of the cross-linked resilin composition is comparable to an orthopedic sole.

In some embodiments, the recombinant resilin composition comprises a rebound resilience from about 40% to about 60% as measured by ASTM D7121.

In some embodiments, the recombinant resilin composition comprises a compressive stress at 25% of about 6 psi to about 8 psi as measured by ASTM D575.

In some embodiments, the recombinant resilin composition does not undergo an elastic to plastic transition below 2 kN of compressive force as measured by a Zwick compression test.

In some embodiments, resilin solid material properties, such as resilience, compressive elastic modulus, tensile elastic modulus, shear modulus, extension to break, maximum tensile strength, hardness, stiffness, and rebound can be tuned based on the solvent used and how the solvent exchange is performed. For example, glycerol can be exchanged into the resilin solid either with a gradient or without, result in different resulting material properties. The concentration of resilin in the resilin solid and the amount of full length resilin as a portion of total resilin can also be adjusted to affect the material properties of the cross-linked resilin solid composition.

In some embodiments, solvent exchange to replace the water-based resilin composition (i.e, a cross-linked resilin composition in an aqueous solvent) with a polar nonaqueous-based resilin composition (i.e., a cross-linked resilin composition in a polar nonaqueous solvent) as described herein resulting in a stiffer material with a similar resilience and a similar elasticity.

In some embodiments, the compositions provided herein comprise at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%; between 10% and 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%; between 20% and 100%, 90%, 80%, 70%, 60%, 50%, 40%, or 30%; between 30% and 100%, 90%, 80%, 70%, 60%, 50%, or 40%; between 40% and 100%, 90%, 80%, 70%, 60%, or 50%; between 50% and 100%, 90%, 80%, 70%, or 60%; between 60% and 100%, 90%, 80%, or 70%; between 70% and 100%, 90%, or 80%; between 80% and 100%, or 90%; or between 90% and 100% by weight of recombinant resilins. The recombinant resilins can be identical recombinant resilins or mixtures of recombinant resilins having at least 2 different amino acid sequences.

In some embodiments, the compositions provided herein comprise at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%; between 10% and 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%; between 20% and 100%, 90%, 80%, 70%, 60%, 50%, 40%, or 30%; between 30% and 100%, 90%, 80%, 70%, 60%, 50%, or 40%; between 40% and 100%, 90%, 80%, 70%, 60%, or 50%; between 50% and 100%, 90%, 80%, 70%, or 60%; between 60% and 100%, 90%, 80%, or 70%; between 70% and 100%, 90%, or 80%; between 80% and 100%, or 90%; or between 90% and 100% by weight of full length recombinant resilins as a portion of total resilin in each composition.

In some embodiments, a recombinant resilin composition is a foam material. In some embodiments, a cross-linked resilin composition is a foam material, e.g., a foamed solid. In some embodiments, the foam material comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% porosity. As described herein, porosity refers to the percentage of the volume of interstices of a material to the volume of its mass. In some embodiments, density of the foam material as described herein is at least ⅓rd the density of a non-foam material, e.g., at least ½, at least ¾th, or at least twice the density of a non-foam cross-linked resilin composition. In some embodiments, average diameter of the bubbles in the foam material as described herein is about 0.01 mm to about 3 mm, e.g., about 0.02 mm to about 1 mm, about 0.05 mm to about 2 mm, about 0.1 mm to about 3 mm, about 0.2 mm to about 4 mm, about 0.5 mm to about 5 mm, about 1 mm to about 1.5 mm, or about 1.5 mm to about 2 mm.

In some embodiments, a recombinant resilin composition comprises one or more layers of foam material. In some embodiments, the one or more layers of foam material are configured in a desired order, e.g. a sequential or alternate configuration, wherein a supporting layer is positioned in between two layers of foam material. The one or more layers of foam material may be positioned in any manner known in the art, e.g., spray drying, injection molding. In some embodiments, the one or more layers of foam material includes one or more void spaces. The topography of each layer and the propensity of the one or more layers of foam material to nest together may differ. In some embodiments, the void spaces may provide flow channels on one or more sides to help guide air flow and provide additional absorbent capacity.

Mechanical Properties

In other embodiments, the compositions provided herein have different properties compared to compositions comprising cross-linked resilins. In some embodiments, the compositions provided herein have similar properties compared to synthetic elastic materials. Non-limited examples of such properties include resilience, compressive elastic modulus, tensile elastic modulus, shear modulus, hardness, rebound, and compression set. Parameters that can be modified to obtain compositions with specific mechanical properties include, for example, the length and/or sequence of the recombinant resilins, the extent and/or type of post-translational modifications of the recombinant resilins, the extent and/or type of cross-linking of the recombinant resilins and the nature of the solvent of the cross-linked resilin composition.

In some embodiments, mechanical properties such as maximum tensile strength, compressive elastic modulus, tensile elastic modulus, shear modulus, extension to break and resilience can be measured using many different types of tensile and compression systems that conduct stress-strain measurements on elastomeric samples. The resulting stress-strain curves, including curves with hysteresis, can be measured in tension or compression. In some embodiments, tension and compression test systems can apply a strain to a sample and measure the resulting force using a load cell. In some embodiments, the mechanical properties can be measured at the macroscopic scale (e.g., using macroscopic compression testers), microscopic, or nanoscopic scale (e.g., using atomic-force microscopy (AFM) or nanoindentation measurements). In some embodiments, the compressive mechanical properties of elastomers can be measured according to the standard ASTM D575-91(2012) Standard Test Methods for Rubber Properties in Compression. Mechanical measurements of elastomers in tension can be performed using ASTM D412-15a Standard Test Methods for Vulcanized Rubber and Thermoplastic Elastomers-Tension. In some embodiments, tear strength of elastomers can be performed using ASTM D624-00 Standard Test Method for Tear Strength of Conventional Vulcanized Rubber and Thermoplastic Elastomers. In some embodiments, mechanical properties of slab, bonded, and molded elastomers can be performed using ASTM D3574-11 Standard Test Methods for Flexible Cellular Materials—Slab, Bonded, and Molded Urethane Foams. In some embodiments, the mechanical properties of elastomers can be measured using ASTM D5992-96(2011) Standard Guide for Dynamic Testing of Vulcanized Rubber and Rubber-Like Materials Using Vibratory Methods.

In some embodiments, the compositions provided herein have a resilience of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95%; from 50% to 100%, 90%, 80%, 70%, or 60%; from 60% to 100%, 90%, 80%, or 70%; from 70% to 100%, 90%, or 80%; from 80% to 100%, or 90%; from 90% to 100%; from 95% to 100%, from 90% to 99%, or from 95% to 99%.

In some embodiments, the compositions provided herein have a compressive elastic modulus of less than 10 MPa, less than 7 MPa, less than 5 MPa, less than 2 MPa, less than 1 MPa, less than 0.5 MPa, or less than 0.1 MPa; from 0.01 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, 1 MPa, 0.5 MPa, or 0.1 MPa; from 0.1 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, 1 MPa, or 0.5 MPa; from 0.5 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, or 1 MPa; from 1 MPa to 10 MPa, 7 MPa, 5 MPa, or 2 MPa; from 2 MPa to 10 MPa, 7 MPa, or 5 MPa; from 5 MPa to 10 MPa, or 7 MPa; or from 7 MPa to 10 MPa. In some embodiments, the compressive elastic modulus of a composition can be measured as defined by the ASTM D575-91(2012) Standard Test Methods for Rubber Properties in Compression.

In some embodiments, the compositions provided herein have a tensile elastic modulus of less than 10 MPa, less than 7 MPa, less than 5 MPa, less than 2 MPa, less than 1 MPa, less than 0.5 MPa, or less than 0.1 MPa; from 0.01 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, 1 MPa, or 0.5 MPa; from 0.5 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, or 1 MPa; from 1 MPa to 10 MPa, 7 MPa, 5 MPa, or 2 MPa; from 2 MPa to 10 MPa, 7 MPa, or 5 MPa; from 5 MPa to 10 MPa, or 7 MPa; or from 7 MPa to 10 MPa.

In some embodiments, the compositions provided herein have a shear modulus of less than 1 MPa, less than 100 kPa, less than 50 kPa, less than 20 kPa, less than 10 kPa, or less than 1 kPa; from 0.1 kPa to 1 MPa, 100 kPa, 50 kPa, 20 kPa, 10 kPa, or 1 kPa; from 1 kPa to 1 MPa, 100 kPa, 50 kPa, 20 kPa, or 10 kPa; from 10 kPa to 1 MPa, 100 kPa, 50 kPa, or 20 kPa; from 20 kPa to 1 MPa, 100 kPa, or 50 kPa; from 50 kPa to 1 MPa, or 100 kPa; or from 100 kPa to 1 MPa.

In some embodiments, the compositions provided herein have an extension to break of greater than 1%, greater than 10%, greater than 50%, greater than 100%, greater than 300%, or greater than 500%; from 1% to 500%, 300%, 100%, 50%, or 10%; from 10% to 500%, 300%, 100%, or 50%; from 50% to 500%, 300%, or 100%; from 100% to 500%, or 300%; or from 300% to 500%.

In some embodiments, the compositions provided herein have a maximum tensile strength of greater than 0.1 kPa, greater than 1 kPa, greater than 2 kPa, greater than 5 kPa, or greater than 10 kPa; from 0.1 kPa to 100 kPa, 10 kPa, 5 kPa, 2 kPa, or 1 kPa; from 1 kPa to 100 kPa, 10 kPa, 5 kPa, or 2 kPa; from 2 kPa to 100 kPa, 10 kPa, or 5 kPa; from 5 kPa to 100 kPa, or 10 kPa; or from 10 kPa to 100 kPa.

In some embodiments, mechanical properties such as hardness and compressive elastic modulus can be measured using indentation and nanoindentation measurement systems. In some embodiments, indentation measurements utilizing a tip to indent the sample to a given amount of strain are used to measure the hardness and compressive elastic modulus of resilin, and the resulting force is measured using a load cell. In some embodiments, different tip shapes can be used including Vickers and Berkovich shaped tips. In some embodiments, the hardness measured by indentation techniques is characterized by the relation, Hardness=(Peak Force)/(Contact Area).

In some embodiments, the hardness in polymers, elastomers, and rubbers can be measured using a durometer. In some embodiments the hardness of an elastomer can be measured using the standard ASTM D2240, which recognizes twelve different durometer scales using combinations of specific spring forces and indentor configurations. The most common scales are the Shore 00, A and D Hardness Scales. Hardness scales range from 0 to 100, where 0 is softer material and 100 is harder material.

In some embodiments, the compositions provided herein have a Shore 00 Hardness of less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, or less than 20; from 10 to 90, 80, 70, 60, 50, 40, 30, or 20; from 20 to 90, 80, 70, 60, 50, 40, or 30; from 30 to 90, 80, 70, 60, 50, or 40; from 40 to 90, 80, 70, 60, or 50; from 50 to 90, 80, 70, or 60; from 60 to 90, 80, or 70; from 70 to 90, or 80; or from 80 to 90. In some embodiments, hardness measurements in resilin are performed according to ASTM D2240.

As used here, the term “rebound” refers to a particular measure of resilience. In some embodiments, rebound can be measured with a number of different tools including pendulum tools and dropped balls. In the pendulum type measurements, RB, commonly called percentage rebound, is determined from the equation:

${RB} = {\frac{\left\lbrack {1 - {\cos \left( {{angle}\mspace{14mu} {of}\mspace{14mu} {rebound}} \right)}} \right\rbrack}{\left\lbrack {1 - {\cos \left( {{originial}\mspace{14mu} {angle}} \right)}} \right\rbrack} \times 100}$

The rebound resilience can be calculated as:

$R = \frac{h}{H}$

where h=apex height of the rebound, and H=initial height. The rebound resilience can also be determined by the measurement of the angle of rebound. Some examples of test methods for determining rebound in elastomers are ASTM D2632-15 and ASTM D7121-05(2012).

In some embodiments, the compositions provided herein have a rebound greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95%; from 50% to 100%, 90%, 80%, 70%, or 60%; from 60% to 100%, 90%, 80%, or 70%; from 70% to 100%, 90%, or 80%; from 80% to 100%, or 90%; from 90% to 100%; from 95% to 100%, from 90% to 99%, or from 95% to 99%. In some embodiments, rebound measurements in resilin are performed according to ASTM D2632-15, or ASTM D7121-05(2012).

As used herein, the term “compression set” refers to a measure of the permanent deformation remaining after an applied force is removed. In some embodiments, compression set can be measured in different ways, including compression set under constant force in air (referred to as Compression Set A), compression set under constant deflection in air (referred to as Compression Set B), and compression set under constant deflection in air considering material hardness (referred to as Compression Set C). Compression Set A (C_(A)) is calculated by the following expression: C_(A)=[(t_(o)−t_(i))/t_(o) ]×100, where to is the original specimen thickness, and t_(i) is the specimen thickness after testing). Compression set B (C_(B)) is given by C_(B)=[(t_(o)−t_(i))/(t_(o)−t_(n))]×100, where t_(o) is the original specimen thickness, t_(i) is the specimen thickness after testing, and t_(o) is the spacer thickness or the specimen thickness during the test. Some examples of test methods for determining compression set in elastomers are ASTM D3574-11 and ASTM D395-16.

In some embodiments, the compositions provided herein have a Compression Set A or a Compression Set B of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95%; from 50% to 100%, 90%, 80%, 70%, or 60%; from 60% to 100%, 90%, 80%, or 70%; from 70% to 100%, 90%, or 80%; from 80% to 100%, or 90%; from 90% to 100%; from 95% to 100%, from 90% to 99%, or from 95% to 99%. In some embodiments, compression set measurements in resilin are performed according to ASTM D3574-11 and ASTM D395-16.

The processing and forming of resilin into products can take many forms for different applications. Accordingly, the compositions provided herein can have any shape and form, including but not limited to gels, porous sponges, films, machinable solids, cast forms, molded forms, and composites.

As used herein, the term “density” refers to the mass of the sample divided by the volume. In some embodiments, the density of an elastomer can be determined using a pycnometer with alcohol in place of water to eliminate air bubbles. In some embodiments, the density of an elastomer can be determined using a hydrostatic method. As used herein, the term “compressed volume density” refers to the ratio of the sample mass to the compressed volume of the sample, where the “compressed volume” is defined as the final equilibrium volume attained by an elastomeric sample when it is subjected to a compressive force sufficient to cause it to flow until it fully conforms to the surrounding shape of the piston-cylinder test chamber enclosure. In some embodiments, the compressed volume density of an elastomer can be determined using a compressed volume densimeter.

In some embodiments, the compositions provided herein have a density or a compressed volume density are from 0.5 mg/cm³ to 2.0 mg/cm³, or from 1.0 mg/cm³ to 1.5 mg/cm³, or from 1.1 mg/cm³ to 1.4 mg/cm³, or from 1.2 mg/cm³ to 1.35 mg/cm³. In some embodiments, he determination of the density or the compressed volume density of elastomers can be performed using ASTM D297-15 Standard Test Methods for Rubber Products—Chemical Analysis.

The compositions provided herein have a number of uses, including but not limited to applications in aerospace, automotive, sporting equipment, vibration isolation, footwear, and clothing among others. Some applications from these categories are listed as non-limiting examples. Due to the desirable elastic efficiency, resilin can be used as an energy storage device (e.g., a rubber band) for storing and recovering mechanical energy. Automobile suspension systems can be improved by application of resilin bushings to keep more tire contact on the road when going over bumps and through potholes at speed. Additionally, there are a number of sporting equipment applications for resilin with differently tuned mechanical properties including cores of golf balls, tennis racket grips, golf club grips, and table tennis paddles.

An application of particular interest is footwear due to the unique properties of resilin compositions provided herein. As an insole or midsole, resilin can improve the comfort and bioefficiency of shoes by cushioning the foot strike and restoring more of the energy from that footstrike as forward momentum. As a midsole, resilin can make up the entire midsole or be encapsulated within another material to complement its properties (e.g., an abrasion or wear resistant material, or a material tuned for traction). The resilin midsole can also contain a plurality of resilin materials with differently tuned mechanical properties that work in concert to provide enhanced performance (e.g., softer heel strike area and firmer arch support).

Vectors, Host Cells, and Fermentations

Further provided herein are vectors encoding recombinant resilins, recombinant host cells comprising such vectors, and fermentations comprising such recombinant host cells and recombinant resilins.

In some embodiments, the vectors provided herein comprise secreted resilin coding sequences, which encode a resilin polypeptide fused at its N-terminus to a secretion signal and optionally at its C-terminus to a tag peptide or polypeptide. In some embodiments, the vectors comprise secreted resilin coding sequences that are codon-optimized for expression in a particular host cell.

Suitable secretion signals are secretion signals that mediate secretion of polypeptides in the recombinant host cells provided herein. Non-limiting examples of suitable secretion signals are the secretion signals of the alpha mating factor (α-MF) of Saccharomyces cerevisiae, acid phosphatase (PH01) of Pichia pastoris, and phytohemagglutinin (PHA-E) from the common bean Phaseolus vulgaris. Additional secretion signals are known in the art, or can be identified by identification of proteins secreted by a host cell followed by genomic analysis of the secreted proteins and identification of the non-translated N-terminal sequences (see, for example, Huang et al. A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures. Appl Microbiol Biotechnol. 2011 April; 90(1):235-47).

The resilins encoded by the secreted resilin coding sequences can be further fused to tag peptides or polypeptides. Non-limiting examples of tag peptides or polypeptides include affinity tags (i.e., peptides or polypeptides that bind to certain agents or matrices), solubilization tags (i.e., peptides or polypeptides that assist in proper folding of proteins and prevent precipitation), chromatography tags (i.e., peptides or polypeptides that alter the chromatographic properties of a protein to afford different resolution across a particular separation techniques), epitope tags (i.e., peptides or polypeptides that are bound by antibodies), fluorescence tags (i.e., peptides or polypeptides that upon excitation with short-wavelength light emit high-wavelength light), chromogenic tags (i.e., peptides or polypeptides that absorb specific segments of the visible light spectrum), enzyme substrate tags (i.e., peptides or polypeptides that are the substrates for specific enzymatic reactions), chemical substrate tags (i.e., peptides or polypeptides that are the substrates for specific chemical modifications), or combinations thereof. Non-limiting examples of suitable affinity tags include maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag, SBP-tag, Strep-tag, and calmodulin-tag. Non-limiting examples of suitable solubility tags include thioredoxin (TRX), poly(NANP), MBP, and GST. Non-limiting examples of chromatography tags include polyanionic amino acids (e.g., FLAG-tag [GDYKDDDDKDYKDDDDKDYKDDDDK (SEQ ID NO: 45)]) and polyglutamate tag. Non-limiting examples of epitope tags include V5-tag, VSV-tag, Myc-tag, HA-tag, E-tag, NE-tag, and FLAG-tag. Non-limiting examples of fluorescence tags include green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), orange fluorescent protein (OFP), red fluorescent protein (RFP), and derivatives thereof. Non-limiting examples of chromogenic tags include non-fluorescent members of the GFP-like family of proteins (e.g., BlitzenBlue, DonnerMagenta; DNA2.0, Neward, Calif.). Non-limiting examples of enzyme substrate tags include peptides or polypeptides comprising a lysine within a sequence suitable for biotinilation (e.g., AviTag, Biotin Carboxyl Carrier Protein [BCCP]). Non-limiting examples of chemical substrate tags include substrates suitable for reaction with FIAsH-EDT2. The fusion of the C-terminal peptide or polypeptide to the resilin can be cleavable (e.g., by TEV protease, thrombin, factor Xa, or enteropeptidase) on non-cleavable.

In some embodiments, the vectors comprise single secreted resilin coding sequences. In other embodiments, the vectors comprise 2 or more (e.g., 3, 4, or 5) secreted resilin coding sequences. In some such embodiments, the secreted resilin coding sequences are identical. In other such embodiments, at least 2 of the secreted resilin coding sequences are not identical. In embodiments in which at least 2 of the secreted resilin coding sequences are not identical, the at least 2 secreted resilin coding sequences can differ from each other in the resilins and/or in the secretion signals and/or the optional tag peptides or polypeptides they encode.

In some embodiments, the vectors comprise promoters that are operably linked to the secreted resilin coding sequences such that they drive the expression of the secreted resilin coding sequences. The promotors can be constitutive promoters or inducible promoters. In some embodiments, induction of the inducible promoter occurs via glucose repression, galactose induction, sucrose induction, phosphate repression, thiamine repression, or methanol induction. Suitable promoters include promoters that mediate expression of proteins in the recombinant host cells provided herein. Non-limiting examples of suitable promoters include the AOX1 promoter, GAP promoter, LAC4-PBI promoter, T7 promoter, TAC promoter, GCW14 promoter, GAL1 promoter, πPL promoter, πPR promoter, beta-lactamase promoter, spa promoter, CYC1 promoter, TDH3 promoter, GPD promoter, TEF1 promoter, ENO2 promoter, PGL1 promoter, SUC2 promoter, ADH1 promoter, ADH2 promoter, HXT7 promoter, PHOS promoter, and CLB1 promoter. Additional promoters that can be used to facilitate expression of the secreted resilin coding sequences are known in the art.

In some embodiments, the vectors comprise terminators that are operably linked to the secreted resilin coding sequences such that they effect termination of transcription of the secreted resilin coding sequences. Suitable terminators include terminators that terminate transcription in the recombinant host cells provided herein. Non-limiting examples of suitable terminators include the AOX1 terminator, PGK1 terminator, and TPS1 terminator. Additional terminators that effect termination of transcription of the secreted resilin coding sequences are known in the art.

In embodiments in which the vectors comprise 2 or more resilin coding sequences, the 2 or more resilin coding sequences can be operably linked to the same promoters and/or terminators or to 2 or more different promoters and/or terminators.

The vectors provided herein can further comprise elements suitable for propagation of the vectors in recombinant host cells. Non-limiting examples of such elements include bacterial origins of replication and selection markers (e.g., antibiotic resistance genes, auxotrophic markers). Bacterial origins of replication and selection markers are known in the art. In some embodiments, the selection marker is a drug resistant marker. A drug resistant maker enables cells to detoxify an exogenously added drug that would otherwise kill the cell. Illustrative examples of drug resistant markers include but are not limited to those for resistance to antibiotics such as ampicillin, tetracycline, kanamycin, bleomycin, streptomycin, hygromycin, neomycin, Zeocin™, and the like. In some embodiments, the selection marker is an auxotrophic marker. An auxotrophic marker allows cells to synthesize an essential component (usually an amino acid) while grown in media that lacks that essential component. Selectable auxotrophic gene sequences include, for example, hisD, which allows growth in histidine-free media in the presence of histidinol. Other selection markers suitable for the vectors of the present invention include a bleomycin-resistance gene, a metallothionein gene, a hygromycin B-phosphotransferase gene, the AURI gene, an adenosine deaminase gene, an aminoglycoside phosphotransferase gene, a dihydrofolate reductase gene, a thymidine kinase gene, and a xanthine-guanine phosphoribosyltransferase gene.

The vectors of the present invention can further comprise targeting sequences that direct integration of the secreted resilin coding sequences to specific locations in the genome of host cells. Non-limiting examples of such targeting sequences include nucleotide sequences that are identical to nucleotide sequences present in the genome of a host cell. In some embodiments, the targeting sequences are identical to repetitive elements in the genome of host cells. In some embodiments, the targeting sequences are identical to transposable elements in the genome of host cells.

In some embodiments, recombinant host cells are provided herein that comprise the vectors described herein. In some embodiments, the vectors are stably integrated within the genome (e.g., a chromosome) of the recombinant host cells, e.g., via homologous recombination or targeted integration. Non-limiting examples of suitable sites for genomic integration include the Ty1 loci in the Saccharomyces cerevisiae genome, the rDNA and HSP82 loci in the Pichia pastoris genome, and transposable elements that have copies scattered throughout the genome of the recombinant host cells. In other embodiments, the vectors are not stably integrated within the genome of the recombinant host cells but rather are extrachromosomal.

Recombinant host cells can be of mammalian, plant, algae, fungi, or microbe origin. Non-limiting examples of suitable fungi include methylotrophic yeast, filamentous yeast, Arxula adeninivorans, Aspergillus niger, Aspergillus niger var. awamori, Aspergillus oryzae, Candida etchellsii, Candida guilliermondii, Candida humilis, Candida lipolytica, Candida pseudotropicalis, Candida utilis, Candida versatilis, Debaryomyces hansenii, Endothia parasitica, Eremothecium ashbyii, Fusarium moniliforme, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Morteirella vinaceae var. raffinoseutilizer, Mucor miehei, Mucor miehei var. Cooney et Emerson, Mucor pusillus Lindt, Penicillium roquefortii, Pichia methanolica, Pichia pastoris (Komagataella phaffii), Pichia (Scheffersomyces) stipitis, Rhizopus niveus, Rhodotorula sp., Saccharomyces bayanus, Saccharomyces beticus, Saccharomyces cerevisiae, Saccharomyces chevaliers, Saccharomyces diastaticus, Saccharomyces ellipsoideus, Saccharomyces exiguus, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces pastorianus, Saccharomyces pombe, Saccharomyces sake, Saccharomyces uvarum, Sporidiobolus johnsonii, Sporidiobolus salmonicolor, Sporobolomyces roseus, Trichoderma reesi, Xanthophyllomyces dendrorhous, Yarrowia lipolytica, Zygosaccharomyces rouxii, and derivatives and crosses thereof.

Non-limiting examples of suitable microbes include Acetobacter suboxydans, Acetobacter xylinum, Actinoplane missouriensis, Arthrospira platensis, Arthrospira maxima, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillusstearothermophilus, Bacillus subtilis, Escherichia coli, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus reuteri, Lactococcus lactis, Lactococcus lactis Lancefield Group N, Leuconostoc citrovorum, Leuconostoc dextranicum, Leuconostoc mesenteroides strain NRRL B-512(F), Micrococcus lysodeikticus, Spirulina, Streptococcus cremoris, Streptococcus lactis, Streptococcus lactis subspecies diacetylactis, Streptococcus thermophilus, Streptomyces chattanoogensis, Streptomyces griseus, Streptomyces natalensis, Streptomyces olivaceus, Streptomyces olivochromogenes, Streptomyces rubiginosus, Xanthomonas campestris, and derivatives and crosses thereof. Additional strains that can be used as recombinant host cells are known in the art. It should be understood that the term “recombinant host cell” is intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but is still included within the scope of the term “recombinant host cell” as used herein.

In some embodiments, the recombinant host cells comprise genetic modifications that improve production of the recombinant resilins provided herein. Non-limiting examples of such genetic modifications include altered promoters, altered kinase activities, altered protein folding activities, altered protein secretion activities, altered gene expression induction pathways, and altered protease activities.

The recombinant host cells provided herein are generated by transforming cells of suitable origin with vectors provided herein. For such transformation, the vectors can be circularized or be linear. Recombinant host cell transformants comprising the vectors can be readily identified, e.g., by virtue of expressing drug resistance or auxotrophic markers encoded by the vectors that permit selection for or against growth of cells, or by other means (e.g., detection of light emitting peptide comprised in vectors, molecular analysis of individual recombinant host cell colonies, e.g., by restriction enzyme mapping, PCR amplification, or sequence analysis of isolated extrachromosomal vectors or chromosomal integration sites).

In some embodiments, the recombinant host cells provided herein can produce high titers of the recombinant resilins provided herein. In some such embodiments, the recombinant host cells produce the recombinant resilins at a rate of greater than 2 mg resilin/g dry cell weight/hour, 4 mg resilin/g dry cell weight/hour, 6 mg resilin/g dry cell weight/hour, 8 mg resilin/g dry cell weight/hour, 10 mg resilin/g dry cell weight/hour, 12 mg resilin/g dry cell weight/hour, 14 mg resilin/g dry cell weight/hour, 16 mg resilin/g dry cell weight/hour, 18 mg resilin/g dry cell weight/hour, 20 mg resilin/g dry cell weight/hour, 25 mg resilin/g dry cell weight/hour, or 30 mg resilin/g dry cell weight/hour; from 2 to 40, 30, 20, 10, or 5 mg resilin/g dry cell weight/hour; from 5 to 40, 30, 20, or 10 mg resilin/g dry cell weight/hour; from 10 to 40, 30, or 20 mg resilin/g dry cell weight/hour; from 20 to 40, or 30 mg resilin/g dry cell weight/hour; or from 30 to 40 mg resilin/g dry cell weight/hour. In other such embodiments, the recombinant host cells secrete the recombinant resilins at a rate of greater than 2 mg resilin/g dry cell weight/hour, 4 mg resilin/g dry cell weight/hour, 6 mg resilin/g dry cell weight/hour, 8 mg resilin/g dry cell weight/hour, 10 mg resilin/g dry cell weight/hour, 12 mg resilin/g dry cell weight/hour, 14 mg resilin/g dry cell weight/hour, 16 mg resilin/g dry cell weight/hour, 18 mg resilin/g dry cell weight/hour, 20 mg resilin/g dry cell weight/hour, 25 mg resilin/g dry cell weight/hour, or 30 mg resilin/g dry cell weight/hour; from 2 to 40, 30, 20, 10, or 5 mg resilin/g dry cell weight/hour; from 5 to 40, 30, 20, or 10 mg resilin/g dry cell weight/hour; from 10 to 40, 30, or 20 mg resilin/g dry cell weight/hour; from 20 to 40, or 30 mg resilin/g dry cell weight/hour; or from 30 to 40 mg resilin/g dry cell weight/hour. The identities of the recombinant resilins produced can be confirmed by HPLC quantification, Western blot analysis, polyacrylamide gel electrophoresis, and 2-dimensional mass spectroscopy (2D-MS/MS) sequence identification.

In some embodiments, the recombinant host cells provided herein have high secreted fractions of the recombinant resilins provided herein. In some such embodiments, the recombinant host cells have secreted fractions of recombinant resilient that is greater than 50%, 60%, 70%, 80%, or 90%; from 50% to 100%, 90%, 80%, 70%, or 60%; from 60% to 100%, 90%, 80%, or 70%; from 70% to 100%, 90%, or 80%; from 90% to 100%, or 90%; or from 90% to 100%.

Production and secretion of recombinant resilins can be influenced by the number of copies of the secreted resilin coding sequences comprised in the recombinant host cells and/or the rate of transcription of the secreted resilin coding sequences comprised in the recombinant host cells. In some embodiments, the recombinant host cells comprise a single secreted resilin coding sequence. In other embodiments, the recombinant host cells comprise 2 or more (e.g., 3, 4, 5, or more) secreted resilin coding sequences. In some embodiments, the recombinant host cells comprise secreted resilin coding sequences that are operably linked to strong promoters. Non-limiting examples of strong promoters include the pGCW14 promoter of Pichia pastoris. In some embodiments, the recombinant host cells comprise secreted resilin coding sequences that are operably linked to medium promoters. Non-limiting examples of such medium promoters include the pGAP promoter of Pichia pastoris. In some embodiments, the recombinant host cells comprise coding sequences encoding resilins under the control of weak promoters.

The fermentations provided herein comprise recombinant host cells described herein and a culture medium suitable for growing the recombinant host cells.

The fermentations are obtained by culturing the recombinant host cells in culture media that provide nutrients needed by the recombinant host cells for cell survival and/or growth, and for secretion of the recombinant resilins. Such culture media typically contain an excess carbon source. Non-limiting examples of suitable carbon sources include monosaccharides, disaccharides, polysaccharides, and combinations thereof. Non-limiting examples of suitable monosaccharides include glucose, galactose, mannose, fructose, ribose, xylose, arabinose, ribose, and combinations thereof. Non-limiting examples of suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of suitable polysaccharides include raffinose, starch, glycogen, glycan, cellulose, chitin, and combinations thereof.

In some embodiments, the fermentations comprise recombinant resilins in amounts of at least 1%, 5%, 10%, 20%, or 30%; from 1% to 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%; from 10% to 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%; from 20% to 100%, 90%, 80%, 70%, 60%, 50%, 40%, or 30%; from 30% to 100%, 90%, 80%, 70%, 60%, 50%, or 40%; from 40% to 100%, 90%, 80%, 70%, 60%, or 50%; from 50% to 100%, 90%, 80%, 70%, or 60%; from 60% to 100%, 90%, 80%, or 70%; from 70% to 100%, 90%, or 80%; from 80% to 100%, or 90%; or from 90% to 100% by weight of the total fermentation.

In some embodiments, the fermentations comprise recombinant resilin in an amount of at least 2 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, or 30 g/L; from 2 g/L to 300 g/L, 200 g/L, 100 g/L, 90 g/L, 80 g/L, 70 g/L, 60 g/L, 50 g/L, 40 g/L, 30 g/L, 20 g/L, or 10 g/L; from 10 g/L to 300 g/L, 200 g/L, 100 g/L, 90 g/L, 80 g/L, 70 g/L, 60 g/L, 50 g/L, 40 g/L, 30 g/L, or 20 g/L; from 20 g/L to 300 g/L, 200 g/L, 100 g/L, 90 g/L, 80 g/L, 70 g/L, 60 g/L, 50 g/L, 40 g/L, or 30 g/L; from 30 g/L to 300 g/L, 200 g/L, 100 g/L, 90 g/L, 80 g/L, 70 g/L, 60 g/L, 50 g/L, or 40 g/L; from 40 g/L to 300 g/L, 200 g/L, 100 g/L, 90 g/L, 80 g/L, 70 g/L, 60 g/L, or 50 g/L; from 50 g/L to 300 g/L, 200 g/L, 100 g/L, 90 g/L, 80 g/L, 70 g/L, or 60 g/L; from 60 g/L to 300 g/L, 200 g/L, 100 g/L, 90 g/L, 80 g/L, or 70 g/L; from 70 g/L to 300 g/L, 200 g/L, 100 g/L, 90 g/L, or 80 g/L; from 80 g/L to 300 g/L, 200 g/L, 100 g/L, or 90 g/L; from 90 g/L to 300 g/L, 200 g/L, or 100 g/L; from 100 g/L to 300 g/L, or 200 g/L; or from 200 g/L to 300 g/L.

Methods of Producing Recombinant Resilin

Further provided herein are methods for the production of the recombinant resilins described herein.

The methods are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1990; Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press, 2003; Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press, 1976; Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press, 1976; Essentials of Glycobiology, Cold Spring Harbor Laboratory Press, 1999.

In some embodiments, a novel method is utilized to secrete resilin extracellularly from a host cell. In some embodiments, the method comprises constructing a vector comprising a secreted resilin coding sequence (step 1001 in FIG. 2), transforming the vector into a host cell (step 1002 in FIG. 2), and then culturing the recombinant host cells to secrete resilin extracellularly (step 1003 in FIG. 2). In some embodiments, the method includes secreting the resilin extracellularly at a rate greater than 2 mg resilin/g dry cell weight/hour, 4 mg resilin/g dry cell weight/hour, 6 mg resilin/g dry cell weight/hour, 8 mg resilin/g dry cell weight/hour, 10 mg resilin/g dry cell weight/hour, 12 mg resilin/g dry cell weight/hour, 14 mg resilin/g dry cell weight/hour, 16 mg resilin/g dry cell weight/hour, 18 mg resilin/g dry cell weight/hour, 20 mg resilin/g dry cell weight/hour, 25 mg resilin/g dry cell weight/hour, or 30 mg resilin/g dry cell weight/hour; from 2 to 40, 30, 20, 10, or 5 mg resilin/g dry cell weight/hour; from 5 to 40, 30, 20, or 10 mg resilin/g dry cell weight/hour; from 10 to 40, 30, or 20 mg resilin/g dry cell weight/hour; from 20 to 40, or 30 mg resilin/g dry cell weight/hour; or from 30 to 40 mg resilin/g dry cell weight/hour. In some embodiments, the secreted resilin is then purified (step 1004 in FIG. 2), and the purified resilin is cross-linked to form an elastomer (step 1005 in FIG. 2). In some embodiments, the methods provided herein comprise the step of transforming cells with vectors provided herein to obtain recombinant host cells provided herein (step 1002 in FIG. 2). Methods for transforming cells with vectors are well-known in the art. Non-limiting examples of such methods include calcium phosphate transfection, dendrimer transfection, liposome transfection (e.g., cationic liposome transfection), cationic polymer transfection, electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, hyrodynamic delivery, gene gun, magnetofection, and viral transduction. One skilled in the art is able to select one or more suitable methods for transforming cells with vectors provided herein based on the knowledge in the art that certain techniques for introducing vectors work better for certain types of cells.

In some embodiments, the methods further comprise the step of culturing the recombinant host cells provided herein in culture media under conditions suitable for obtaining the fermentations provided herein (step 1003 in FIG. 2). In some embodiments, the conditions and culture media are suitable to facilitate secretion of the recombinant proteins from the recombinant host cells into the culture media. Suitable culture media for use in these methods are known in the art, as are suitable culture conditions. Exemplary details of culturing yeast host cells are described in Idiris et al., Appl. Microbiol. Biotechnol. 86:403-417, 2010; Zhang et al., Biotechnol. Bioprocess. Eng. 5:275-287, 2000; Zhu, Biotechnol. Adv. 30:1158-1170, 2012; Li et al., MAbs 2:466-477, 2010.

In some embodiments, the methods further comprise the step of purifying secreted recombinant resilins from the fermentations provided herein to obtain the recombinant resilins provided herein (step 1004 in FIG. 2). Purification can occur by a variety of methods known in the art for purifying secreted proteins from fermentations. Common steps in such methods include centrifugation (to remove cells) followed by precipitation of the proteins using precipitants or other suitable cosmotropes (e.g., ammonium sulfate). The precipitated protein can then be separated from the supernatant by centrifugation, and resuspended in a solvent (e.g., phosphate buffered saline [PBS]). The suspended protein can be dialyzed to remove the dissolved salts. Additionally, the dialyzed protein can be heated to denature other proteins, and the denatured proteins can be removed by centrifugation. Optionally, the purified recombinant resilins can be coacervated.

In various embodiments, methods of purifying the secreted recombinant proteins from the fermentation can include various centrifugation steps in conjunction with solubilizing protein in a whole cell broth or cell pellet with known chaotropes such as urea or guanidine thiocyanate.

In some embodiments, recombinant resilin protein can be purified by centrifuging a whole cell broth to produce a first pellet of cells and a first supernatant, and extracting the first supernatant to produce a clear cell broth. The first supernatant is then precipitated using ammonium sulfate and centrifuged to produce a second pellet and second supernatant which was discarded. The second pellet is then re-suspended in PBS for dialysis. In some embodiments, the dialyzed solution is then subject to high temperature to denature proteins that are less stable than the target recombinant protein. Then, denatured proteins are removed by centrifuging the dialyzed and denatured solution to produce a third pellet and third supernatant. The third supernatant is retained from the denatured solution, then coacervated by chilling the third supernatant to induce a phase separation into a dense lower layer containing the target recombinant resilin protein and an upper layer. Samples purified by this method can be referred to as “CCB” samples. In some embodiments, multiple coacervations are performed by retaining the lower layer and incubating the lower layer at a lower temperature to induce further phase separation.

In some embodiments, recombinant resilin protein can be purified by centrifuging a whole cell broth to produce a first pellet of cells and protein proximal to the cells (e.g. adherent to the cells, on the surface of the cells) and/or insoluble protein (e.g. protein aggregates) and first supernatant, then discarding the first supernatant to obtain the first pellet. The first pellet can then be re-suspended in guanidine thiocyanate to solubilize recombinant resilin protein. The re-suspension is then centrifuged again produce a second pellet and a second supernatant. The second supernatant is then dialyzed against PBS and subject to high temperature in order to denature proteins other than recombinant resilin and centrifuged to produce a third pellet and third supernatant. In some embodiments, the third supernatant is then subject to coacervation by chilling to yield phase separation into a dense lower layer containing recombinant resilin and an upper layer. Samples purified by this method can be referred to as “gel layer” samples. In some embodiments, multiple coacervations are performed by retaining the lower layer and incubating the lower layer at a lower temperature to induce further phase separation.

In some embodiments, recombinant resilin protein can be purified by centrifuging a whole cell broth to produce a pellet and supernatant, then discarding the supernatant to obtain a pellet of cells and protein proximal to the cells (e.g. adherent to the cells, on the surface of the cells) and/or insoluble protein (e.g. protein aggregates). The pellet of cells is then re-suspended in guanidine thiocyanate to solubilize the protein that was proximal to the cells. The re-suspension is centrifuged again produce a second pellet of cells and a second supernatant. The second supernatant is then precipitated with ammonium sulfate and centrifuged to produce a third pellet and third supernatant. The third pellet is then suspended in guanidine thiocyanate, then dialyzed against PBS and subject to high temperature to denature proteins other than recombinant resilin and centrifuged to produce a fourth supernatant and fourth pellet. The fourth supernatant is then subject to coacervation by chilling to yield phase separation. Samples purified by this method can be referred to as “gel layer precipitated” samples.

In some embodiments, recombinant resilin protein can be purified by adding urea to a whole cell broth to solubilize the protein, then centrifuging the whole cell broth to produce a first pellet and first supernatant. The first supernatant is then precipitated using ammonium sulfate and centrifuged to produce a second pellet and second supernatant. The second supernatant is discarded and the second pellet is then re-suspended in guanidine thiocyanate and dialyzed against PBS, then subject to high temperature in order to denature proteins other than recombinant resilin and centrifuged again to produce a third pellet and a third supernatant. The third supernatant is then coacervated by chilling the third supernatant to induce a phase separation into a dense lower layer containing recombinant resilin and an upper layer. Samples purified by this method can be referred to as “Urea WCBE” samples.

In some embodiments, recombinant resilin protein can be purified by centrifuging a whole cell broth to produce a first pellet and first supernatant, then discarding the first supernatant to obtain a first pellet of cells and protein proximal to the cells (e.g. adherent to the cells, on the surface of the cells) and/or insoluble protein (e.g. protein aggregates). The first pellet of cells is then re-suspended in guanidine thiocyanate to solubilize the protein. The re-suspension is centrifuged again to produce a second pellet of cells and a second supernatant. The second supernatant is then dialyzed against PBS and then centrifuged to produce a heavy phase of protein, a light phase of supernatant and a film separating the heavy phase from the light phase. The heavy phase of protein is then isolated by discarding the light phase and the film. Samples purified by this method can be referred to as “Dense layer” samples.

In some embodiments, the recombinant resilin composition is a foam material. In some embodiments, a method of preparing the recombinant resilin foam, comprises: providing a cross-linked recombinant resilin solid composition in an aqueous solvent; exchanging said aqueous solvent with a polar nonaqueous solvent; and introducing one or more bubbles to the cross-linked recombinant resilin solid composition. Any method of introducing bubbles known in the art may be used herein. For instance, methods of introducing bubbles include, but are not limited to, vortexing, mixing, adding yeast, and chemical reactions. In some embodiments, the introducing the one or more bubbles may occur at the same time the cross-linked recombinant resilin solid composition is provided. In some embodiments, the introducing the one or more bubbles may occur after the cross-linked recombinant resilin solid composition is provided.

Blowing agents typically are introduced into polymeric material to make polymer foams. According to one embodiment, a chemical blowing agent is mixed with a polymer. The chemical blowing agent undergoes a chemical reaction in the polymeric material, typically under conditions in which the polymer is molten, causing formation of a gas.

Chemical blowing agents generally are low molecular weight organic compounds that decompose at a particular temperature and release a gas such as nitrogen, carbon dioxide, or carbon monoxide.

Exemplary chemical blowing agents include, but are not limited to, sodium bicarbonate, potassium bicarbonate, ammonium, azodicarbonamide, isocyanate, hydrazine, isopropanol, 5-phenyltetrazole, triazole, 4,4′oxybis(benzenesulfonyl hydrazide) (OBSH), trihydrazine triazine (THT), hydrogen phosphate, tartaric acid, citric acid, and toluenesulphonyl semicarbazide (TSS).

In some embodiments, foaming agents, thickeners, and/or hardeners are added to the recombinant resilin solid. Exemplary foaming agents include, but are not limited to, xanthan gum, sodium dodecyl sulfate, ammonium lauryl sulfate, bovine serum albumin. Exemplary thickeners include, but are not limited to, fumed silica and xanthan gum. Exemplary hardeners include, but are not limited to, aliphatic polyamine, fatty polyamides, aromatic polyamine hardeners, anhydride hardeners, boron trifluoride hardeners, and curing agents (dicyandiamide).

According to another embodiment, a physical blowing agent, i.e., a fluid that is a gas under ambient conditions, is injected into a molten polymeric stream to form a mixture. The mixture is subjected to a pressure drop, causing the blowing agent to expand and form bubbles (cells) in the polymer. In some embodiments, the pressure required is about 500 psi to about 2000 psi, e.g., about 600 psi to about 1000 psi, about 700 psi to about 1500 psi, and about 800 psi to about 2000 psi. In some embodiments, the pressure required is about 500 psi.

Exemplary physical blowing agents include, but are not limited to, chlorofluorocarbon (CFC), dissolved nitrogen, N₂, CH₄, H₂, CO₂, Ar, pentane, isopentane, hexane, methylene dichloride, and dichlorotetra-fluoroethane.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B(1992).

Example 1: Production of Recombinant Resilin

Pichia pastoris recombinant host cells that secrete recombinant resilin were generated by transforming a HIS+ derivative of GS115 (NRRL Y15851) Pichia pastoris (Komagataella phaffii) with vectors comprising secreted resilin coding sequences.

The vectors each comprised 3 resilin coding sequences fused in frame to an N-terminal secretion signal (alpha mating factor leader and pro sequence), and in some instances a C-terminal 3×FLAG tag (SEQ ID NO: 45) (see FIG. 3). Each of the secreted resilin coding sequences was flanked by a promoter (pGCW14) and a terminator (tAOX1 pA signal). The vectors further comprised a targeting region that can direct integration of the 3 secreted resilin coding sequences to the HSP82 locus of the Pichia pastoris genome, dominant resistance markers for selection of bacterial and yeast transformants, as well as a bacterial origin of replication.

The resilin coding sequences were obtained from scientific literature and from searching public sequence databases. The nucleotide sequences were translated into amino acid sequences and then codon-optimized. Both full length and truncated resilin sequences were chosen. Selected secreted resilin coding sequences are listed in Table 1.

TABLE 1 Exemplary full-length and truncated resilin amino acid sequences and recombinant host strains Short Amino Acid With FLAG tag Without FLAG tag Species Type Name SEQ ID NO: Plasmid Strain Plasmid Strain Drosophila Full length Ds_ACB 1 RMp4830 RMs1209 RMp4842 RMs1221 sechellia Drosophila A repeats + Ds_AC 2 RMp4831 RMs1210 RMp4843 RMsl222 sechellia Chitin binding domain Drosophila A repeats Ds_A 3 RMp4832 RMs1211 RMp4844 RMs1223 sechellia only Acromyrmex A repeats Ae_A 4 RMp4833 RMS1212 RMp4845 RMs1224 echinatior only Aeshna sp. B repeats As_B 5 RMp4834 RMs1213 RMp4846 RMs1225 only Aeshna sp. Full length As_ACB 6 RMp4835 RMs1214 RMp4847 RMS1226 Haematobia A repeats Hi_A 7 RMp4836 RMs1215 RMp4848 RMS1227 irritans only Haematobia Full length Hi_ACB 8 RMp4837 RMs1216 RMp4849 RMS1228 irritans Ctenocephalides A repeats Cf_A 9 RMp4838 RMs1217 RMp4850 RMs1229 felis only Ctenocephalides B repeats Cf_B 10 RMp4839 RMs1218 RMp4851 RMs1230 felis only Bombus A repeats Bt_A 11 RMp4840 RMs1219 RMp4852 RMs1231 terrestris only Tribolium A repeats Tc_A 12 RMp4841 RMs1220 RMp4853 RMs1232 castaneum only

The vectors were transformed into Pichia pastoris using electroporation to generate host strains comprising 3 integrated copies of each secreted resilin coding sequence. Transformants were plated on YPD agar plates supplemented with an antibiotic, and incubated for 48 hours at 30° C.

Clones from each final transformation were inoculated into 400 μL of Buffered Glycerol-complex Medium (BMGY) in 96-well blocks, and incubated for 24 hours at 30° C. with agitation at 1,000 rpm. A sample was removed, the recombinant host cells were pelleted via centrifugation, and the supernatant was recovered and run on a SDS-PAGE gel for analysis of resilin content via Coomassie gel and Western blot analysis (for polypeptides comprising the 3×FLAG tag). For FLAG-tagged proteins, the remaining cultures were used to inoculate minimal media cultures in duplicate for ELISA measurements. One duplicate was pelleted and the supernatant was measured directly. The second duplicate was extracted with guanidine thiocyanate and both the intra- and extra-cellular fractions were measured.

Recombinant resilin from numerous species expressed successfully in the Pichia pastoris recombinant host cells. Recombinant host cells secreted up to 90% of the recombinant resilin produced.

SEQ ID NO: 49 of the sequence listing provides the full-length Drosophila sechellia resilin sequence (Ds_ACB) that is expressed along with signal sequences that are later cleaved. The first sequence (italics), is an alpha mating factor precursor protein signal sequence (SEQ ID NO: 46) that is cleaved twice after transcription by a signal peptidase followed by cleavage with Kex2. The second sequence (bold) is an EAEA repeat that is cleaved by Ste13 (SEQ ID NO: 47). The third sequence (lower case) corresponds to Drosophila sechellia full-length resilin (SEQ ID NO 1). The fourth sequence (bold and italicized) corresponds to a linker sequence (SEQ ID NO: 46). The fifth sequence (underlined) corresponds to the 3×FLAG tag (SEQ ID NO: 45).

Edman sequencing confirmed that the N-terminus of the protein sequences at the approximately 110 kDa band corresponded to the full-length length Drosophila sechellia resilin sequence. Specifically, the N-terminus sequencing showed that the N-terminus either corresponded to “EAEA” or “GRPE”, respectively the full-length Drosophila sechellia resilin sequence with or without the EAEA repeat.

Example 2: Purification of Recombinant Resilin

Non-FLAG-tagged Ds_ACB and Ae_A recombinant resilin polypeptides were purified as follows: Strains RMs1221 (expressing Ds_ACB) and RMs1224 (expressing Ae_A) were grown in 500 mL of BMGY in flasks for 48 hours at 30° C. with agitation at 300 rpm.

The protocol for purification was adapted from Lyons et al. (2007). Cells were pelleted by centrifugation, and supernatants were collected. Proteins were precipitated by addition of ammonium sulfate. The precipitated proteins were resuspended in a small volume of phosphate buffered saline (PBS), and the resuspended samples were dialyzed against PBS to remove salts. The dialyzed samples were then heated to denature native proteins, and denatured proteins were removed by centrifugation. The retained supernatants contained the purified resilin polypeptides. Optionally, the retained supernatants were chilled, which caused coacervation, resulting in a concentrated lower phase and dilute upper phase.

A second set of samples was prepared by centrifuging a whole cell broth to produce a first pellet of cells and protein proximal to the cells (e.g. adherent to the cells, on the surface of the cells) and/or insoluble protein (e.g. protein aggregates) and first supernatant, then discarding the first supernatant to obtain the first pellet. The first pellet was re-suspended in guanidine thiocyanate to solubilize Ds_ACB. The re-suspension was centrifuged again produce a second pellet and a second supernatant. The second supernatant was then dialyzed against PBS and subject to high temperature in order to denature proteins other than Ds_ACB and centrifuged to produce a third pellet and third supernatant. The third supernatant was subject to coacervation by chilling to yield phase separation into a dense lower layer containing Ds_ACB and an upper layer. This is also referred to herein as gel layer separation, or coacervation samples. In some gel layer samples, multiple coacervations were performed by retaining the lower layer and incubating the lower layer at a lower temperature to induce further phase separation.

TABLE 2 Stability of Cross-linked Resilin 110 kDa Sample band? Degradation Time as solid A Yes Substantial 13 days B No Substantial 8 days C No Substantial 6 days D No Substantial 6 days E Yes Minimal N/A F Yes Minimal 27 days G No Substantial 8 days H Yes Minimal 15 days I Yes Substantial 15 days J No Substantial 6 days K Yes Minimal 13 days L Yes Minimal 13 days

Example 3: Cross-Linking of Purified, Secreted, Recombinant Resilin

Purified resilin was cross-linked via one of three methods: photo cross-linking (adapted from Elvin et al. 2005), enzymatic cross-linking (adapted from Qin et al. 2009), or the method described herein (i.e., exposure to ammonium persulfate and heat).

Photo Cross-Linking

For photo cross-linking, resilin protein was mixed with ammonium persulfate and tris (bipyridine) ruthenium (II) ([Ru(bpy)3]2+). The mixture was exposed to bright white light, after which the mixture formed a rubbery solid.

Specifically, a 10 mM Ru (II) photocatalyst (CAS 50525-27-4) in water and 100 mM ammonium persulfate in water stock solutions were prepared. Purified resilin powder was dissolved in phosphate buffered saline to a concentration between 20-30 wt % resilin to prepare the resilin stock solution.

2 μL of the 10 mM Ru (II) photocatalyst solution and 2 μL of the 100 mM ammonium persulfate was added to and mixed with 10 μL of resilin stock solution to form the photo cross-linking solution. Bubbles were removed from the photo cross-linking solution by centrifugation, and the photo cross-linking solution was poured into a mold.

The photo cross-linking solution was then exposed to white light (White LED floodlight, Zochlon USPT100W 110V) for 30 seconds to 1 minute.

Enzymatic Cross-Linking

For enzymatic cross-linking, resilin protein was mixed with horseradish peroxidase (HRP) and hydrogen peroxide. The mixture was incubated at 37° C., after which the mixture formed a rubbery solid.

Specifically, a 10 mg/mL stock of horse radish peroxidase (type 1, P8125 Sigma) in water (HRP stock) and a 100 mM H₂O₂ stock in water. Purified resilin powder was dissolved in phosphate buffered saline to a concentration between 20-30 wt % resilin to prepare the resilin stock solution.

6 μL of HRP stock was added to and mixed with 21 μL of the resilin stock solution. 3 uL H₂O₂ stock was then added and mixed by vortexing to form the enzymatic cross-linking solution. Bubbles were removed from the enzymatic cross-linking solution by centrifugation, and the enzymatic cross-linking solution was poured into a mold while maintaining the solution at 4° C. To perform cross-linking, the enzymatic cross-linking solution was incubated for 15 minutes at 37° C. The resulting cross-linked resilin composition includes an active enzyme covalently bound to the resilin solid.

Persulfate and Heat Cross-Linking

Purified resilin was cross-linked using ammonium persulfate and heat.

Specifically, purified resilin powder was dissolved in phosphate buffered saline to a concentration between 20-30 wt % resilin to prepare the resilin stock solution. 4 μL of n 200 mM ammonium persulfate in water was mixed with 18 μL of the resilin stock solution to a final concentration of 36 mM ammonium persulfate. Bubbles were removed from the persulfate cross-linking solution by centrifugation, and the persulfate cross-linking solution was poured into a mold.

To perform cross-linking, the persulfate cross-linking solution was incubated at 80° C. for 2.5 hours in a sealed humid environment. The resulting cross-linked resilin composition does not leave an active catalyst behind in the resilin solid and results in a composition with a less degraded protein.

Degradation of HRP Cross-Linked Compositions

The stability of the cross-linked samples was assessed over time by determining the duration each cross-linked samples remained a solid through daily observation. Table 3 shows the time as a solid for each cross-linked sample prepared via photo cross-linking (Ru+hv), enzymatic cross-linking (HRP), and cross-linking via ammonium persulfate and heat (AP+heat). As shown in Table 3, at room temperature, the resilin compositions cross-linked via photo cross-linking or via ammonium persulfate had a much longer duration of stability than resilin compositions cross-linked via enzymatic cross-linking.

TABLE 3 Degradation of resilin compositions cross-linked by different cross-linking procedures Incubation Observed Melt Resilin # Crosslinking Washing? Temp. (° C.) Time (days) 1967 I-8 HRP N RT 8 1967 I-8 HRP N RT 8 1729 I-10 HRP N RT 8 1729 I-10 HRP N RT 10 1967 I-8 Ru + hv N RT >92 1967 I-8 Ru + hv N RT >92 1729 I-10 AP + heat N RT >91 1967 I-8 AP + heat N RT >91

The total degraded resilin from ammonium persulfate cross-linked resilin compositions (AP) and enzymatically cross-linked resilin compositions (HRP) as measured by ELISA is shown in FIG. 6. Increased degradation and secretion of resilin fragments from cross-linked resilin compositions is observed in the HRP samples as compared to AP samples.

This data shows that ammonium persulfate cross-linking is preferred over enzymatic cross-linking for preparation of stable resilin compositions. For enzymatically cross-linked resilin compositions, the cross-linking enzyme remains in the composition and results in degradation of the composition.

Example 4: Stability of Cross-Linked Full-Length Resilin Vs. Resilin Mixture

Resilin samples generated with varying levels of degradation products and full-length resilin were subject to enzymatic cross-linking as described above. The stability of the cross-linked samples was assessed over time by determining the duration each cross-linked samples remained a solid through daily observation. Table 4 shows the time as a solid for each cross-linked sample. As shown in Table 4, samples comprising full-length resilin had a longer duration of stability than the samples that did not comprise full-length resilin.

TABLE 4 Stability of cross-linked resilin 110 kDa Sample band? Degradation Time as solid A Yes Substantial 13 days B No Substantial 8 days C No Substantial 6 days D No Substantial 6 days E Yes Minimal N/A F Yes Minimal 27 days G No Substantial 8 days H Yes Minimal 15 days I Yes Substantial 15 days J No Substantial 6 days K Yes Minimal 13 days L Yes Minimal 13 days

Example 5: Resilin Foam

Crosslinking resilin in the presence of a thickening additive, e.g., fumed silica, and sodium bicarbonate resulted in a foamed resilin with a relatively uniform distribution of bubbles. Altering the amount of fumed silica impacted average bubble size, giving us a lever to tune foamed resilin solids. The results are shown in Table 5.

TABLE 5 Data on resilin foam Property Diameter of bubbles of resilin foam 0.2-2 mm with 5 wt % fumed silica (mm) Diameter of bubbles of resilin foam 0.05-0.2 mm with 10 wt % fumed silica (mm) Amount of sodium bicarbonate (% wt) 0.2-2% wt Amount of thickener (% wt) 0-10% wt

A solution of resilin was prepared. 0.81 g of resilin was dissolved in 2.19 mL PBS at pH of 7.4. A foaming agent was added and the solution was vortexed to introduce bubbles and crosslinked via heat or light. Fifty mg of xantham gum was added to the solution followed by 0.65 mL of 225 mM ammonium persulfate. Xanthum gum was used as the foaming agent because it also increased viscosity which aided in trapping the bubbles in solution. The solution was then vortexed and heated at 80° C. for 3.5 h.

In some samples, ruthenium (II) and white light were used to crosslink the resilin. In such experiments, 0.405 g resilin lot 3 was dissolved in 1.10 mL PBS. Then, 0.3 mL of 100 mM ammonium persulfate and 0.3 mL of 10 mM Ru(II) catalyst. In other samples, 0.405 g resilin lot 3 was dissolved in 1.10 mL PBS. Then, 25 mg of xantham gum was added, followed by 0.3 mL of 100 mM ammonium persulfate and 0.3 mL of 10 mM Ru(II) catalyst.

In some samples, sodium bicarbonate was used as a chemical blowing agent. Sodium bicarbonate was added to the resilin solution and crosslinked. A thickener, fumed silica, was added in some samples. Too much sodium bicarbonate (33 mg/mL or more) inhibited gelation. Using between 6-20 mg/mL of sodium bicarbonate to resilin solution resulted in resilin solids after 3.5 hours at 80° C. with large bubbles. Adding a thickener, fumed silica, to between 4-10 wt %, resulted in a resilin foam with an even distribution of bubbles (FIGS. 7 and 8). Too little fumed silica resulted in a half-foamed resilin solid and adding more than 5 wt % fumed silica resulted in a foamed resilin with smaller bubbles (FIG. 7). The diameter of bubbles of resilin foam with 5 wt % fumed silica was 0.2 to 2 mm. The diameter of bubbles of resilin foam with 10 wt % fumed silica was 0.05 to 0.2 mm.

Specifically, 0.27 g of resilin lot 3 was dissolved in 0.73 mL PBS at pH of 7.4. Then, 37, 18, 10 or 6 mg of sodium bicarbonate was added to the solution followed by 0.22 mL of 225 mM ammonium persulfate and between 0-10 wt % fumed silica. The solution was vortexed and heated at 80° C. for 3.5 h. Experiments were performed in a capped 2 mL tube.

In some samples, an ISCO pump was used to introduce dissolved nitrogen at either 1600 psi or 500 psi while crosslinking the resilin at 83° C. Crosslinking at these pressures proceeded at a slower rate than crosslinking at atmospheric pressure, likely due to the increased amounts of dissolved oxygen.

Specifically, 1.1 mL solution of 225 or 550 mM ammonium persulfate in water was added to a 5 mL solution of 27 wt % resilin in PBS. This solution was then centrifuged for 5 min at 7197 rcf and added to the ISCO pump. The pump was then hooked up to house nitrogen and the ISCO pump was set to its maximum volume of 266 mL, purged with nitrogen for a 3 minutes and then sealed. Then, the pump was either set to 500 psi or 1600 psi and heated for between 2-6 h at 83° C. After releasing the pressure, the resilin was heated for an additional 1-2 hours at 83° C.

Example 6: Solvent Exchange and Comparison of Cross-Linked Resilin in Aqueous Solvent Vs. Polar Nonaqueous Solvent

As described in detail below, a cross-linked mixture of recombinant resilin in an aqueous solvent was treated with a solvent exchange to replace the solvent with a polar nonaqueous solvent to form a composition with material properties better suited for certain applications.

A resilin powder comprising 19 mass % full-length resilin as measured via SEC (size exclusion chromatography), with the rest of the powder comprising partially degraded resilin, was prepared.

The resilin powder was then dissolved in 1× phosphate buffered saline (PBS), the reported weight percent of resilin is weight by weight (w/w). A 1 g solution of 27 wt % resilin is prepared by adding 0.27 g of resilin to 0.73 g of PBS solution. The resilin solution was then cross-linked in a mold to form a water-based resilin solid composition. The resilin solid formed was a 15 mm diameter×5 mm height disc.

To generate a glycerol-based resilin solid, the water-based solid is exchanged in 20× its volume of 60% glycerol (60:40 glycerol:water (v:v)) at 60 deg C. in a closed container for 16 h. Then, the solid is exchanged in 20× its volume of glycerol at 60 deg C. in an unsealed container for 16 h.

Samples of water-based and glycerol-based resilin solids were stored unsealed for 1 week at room temperature. As shown in FIG. 9, water-based resilin solids dehydrated after only 1 week, while glycerol-based resilin solids retained their original shape without any visible degradation or dehydration.

Example 7: Solvent Exchange and Comparison of Properties of Multiple Polar Nonaqueous Resilin Solvents

Resilin solids in polar nonaqueous solvents were prepared from cross-linked resilin in 1× phosphate buffered saline (PBS) as follows:

Resilin solid in propylene glycol solvent was prepared by exchanging the resilin solid in 20× its volume of propylene glycol at 60 deg C. in an unsealed container for 16 h or for 2 days (i.e., 2 d. inc.).

Resilin solid in 60-100% glycerol solvent was prepared by exchanging the resilin solid in 20× its volume of 60% glycerol (60:40 glycerol:water (v:v)) at 60 deg C. in a closed container for 16 h. Then, the solid is exchanged in 20× its volume of glycerol at 60 deg C. in an unsealed container for 16 h.

Resilin solid in 100% glycerol solvent was prepared by exchanging the resilin solid in 20× its volume of glycerol at 60 deg C. in an unsealed container for 16 h or for 2 days (i.e., 2 d. inc.).

Resilin solid in ethylene glycol solvent was prepared by exchanging the resilin solid in 20× its volume of ethylene glycol at 60 deg C. in an unsealed container for 16 h.

Resilin solid in propylene glycol solvent was prepared by exchanging the resilin solid in 20× its volume of propylene glycol at 60 deg C. in an unsealed container for 16 h.

To form a resilin solid based on a purification via coacervation, a 27 wt % (w/w) resilin solution was incubated at −5 deg C. and a liquid-liquid phase separation occurred. The bottom coacervate layer was isolated and ammonium persulfate (APS) powder was mixed into the coacervate to a final concentration of 36 mM APS. This solution was then crosslinked at 80 deg C for 2.5 h. The solid was then exchanged following the typical solvent exchange procedure.

Unless otherwise stated, the resilin powder used is 19 mass % full-length as measured via size-exclusion chromatography (SEC) and the rest of the powder is composed of partially degraded resilin. The 27 wt % “full-length resilin” sample was prepared from resilin powder that is 87 mass % full-length monomeric resilin as measured via SEC.

To characterize the relative amount of the predominant species of resilin in a resilin composition using SEC, resilin powder was dissolved in 5M Guanidine Thiocyanate and injected onto a Yarra SEC-3000 SEC-HPLC column to separate constituents on the basis of molecular weight. Refractive index was used as the detection modality. BSA was used as a general protein standard with the assumption that >90% of all proteins demonstrate do/dc values (the response factor of refractive index) within ˜7% of each other. Poly(ethylene oxide) was used as a retention time standard, and a BSA calibrator was used as a check standard to ensure consistent performance of the method. A range corresponding to resilin was assessed from 60-40 kDa; higher peaks observed which may correspond to aggregate or polymerized resilin were not included in this quantification. The relative percentage of this resilin peak was reported as mass % and area % to determine the amount of full length resilin in a composition as a portion of total resilin.

A Malvern Kinexus Rheometer RNX2110 was then used to generate stress strain curves for each of the compositions produced as described above. The following protocol was applied at a temperature of 22 deg C.: Step 1: Initial Gap—6.5 mm; Step 2: Descent to final Gap. Steps 1 and 2 were repeated 5×. Values of elastic modulus as measured by the rheometer are relative only, and cannot be compared to materials tested under different conditions or with different instruments.

Stress-strain curves were generated plotting engineering stress vs. engineering strain. Engineering stress is calculated by the following formula: Engineering stress=(Normal Force (N)/A (mm²) of resilin solid in contact with rheometer geometry)×10⁶. Engineering strain is the absolute value of compressive strain=ABS((current compressed height of resilin solid (mm)−height of uncompressed solid (mm))/ht. of uncompressed solid (mm))*100.

FIG. 10A shows a stress-strain curve for 27 wt % cross-linked resilin in 60-100% glycerol. FIG. 10B shows a stress-strain curve for 27 wt % cross-linked resilin in propylene glycol. FIG. 10C shows a stress-strain curve for 27 wt % full length cross-linked resilin in propylene glycol. For comparative purposes, rheometry data was also generated from Dr. Sholl's insert.

As shown in FIG. 11, the stress-strain curve of foamed resilin was shaped more like a hockey stick. This is a result of the air pockets in the foamed resilin, which are compressed first, followed by compression of the resilin material.

From the rheometry data, relative values of elastic modulus and resilience were determined as shown in Table 5. Relative Resilience (%) was calculated by taking the area under the load curve and dividing it by the area under the unload curve on the engineering stress vs strain plot and multiplying this value by 100. Relative Elastic modulus was calculated by taking the slope of the load curve from between 10 and 20% absolute compressive strain.

As shown, the exchange of cross-linked resilin solvent from an aqueous water-based solvent to a polar nonaqueous solvent resulted in a stiffer material with a similar resilience and a similar elasticity. All cross-linked resilin compositions were elastic under the conditions tested (<20 N Force).

A rheomoter was also used to measure resilience for a 5% by weight foamed resilin produced as described in Example 5 with a fumed silica as the blower in 100% glycerol solvent. The results are also shown in Table 6.

TABLE 6 Relative values of elastic modulus and resilience for cross-linked resilin in different solvents Elastic Solid Sample Sample Resilience Modulus Description ID (%) (kPa) Dr Scholl's insert, 20 mm diameter 92 1.3 20 wt % resilin, propylene glycol A 59 4.9 27 wt % resilin, propylene glycol B 64 3.9 40 wt % resilin, propylene glycol C 59 6.6 Resilin Coacervate, propylene glycol D 48 6.8 27 wt % full-length resilin, E 34 8.5 propylene glycol 27 wt % resilin, 60-100% glycerol F 94 0.9 27 wt % resilin, 100% glycerol H 70 3.8 27 wt % resilin, ethylene glycol I 90 2.2 27 wt % resilin, water-based solid J 86 1 5 wt % foamed resilin (fumed silica), 35 100% glycerol

The relative values of resilience and elastic modulus from Table 6 were plotted as shown in FIG. 12. This plot shows the relationship between resilience and elastic modulus when tuning the cross-linked resilin properties via solvent exchange.

A rheometer was also used to measure relative resilience and elastic modulus for cross-linked resilin solids in propylene glycol prepared separately. The results are shown in Table 7.

TABLE 7 Rheometer data for identically prepared propylene glycol solids Elastic Solid Sample Resilience Modulus Description (%) (KPa) 27 wt % resilin, propylene glycol, solid 1 64 3.9 27 wt % resilin, propylene glycol, solid 2 59 5.8 27 wt % resilin, propylene glycol, solid 3 63 4.3

A rheometer was also used to measure relative resilience and elastic modulus for cross-linked resilin solids in different solvents through repeated applications of force to determine the effects of the repeated applications of force on these parameters. The results are shown in Table 8.

TABLE 8 Rheometer data: Resilience and modulus data from multiple compressions Elastic Solid Sample Resilience Modulus Description (%) (KPa) Dr. Scholl's insesrt, 20 mm diameter, 92 1.3 1^(st) compression Dr. Scholl's insesrt, 20 mm diameter, 92 1.3 2^(nd) compression Dr. Scholl's insesrt, 20 mm diameter, 95 1.3 3^(rd) compression 27 wt % resilin, 60-100% glycerol, 94 0.9 1^(st) compression 27 wt % resilin, 60-100% glycerol, 88 0.9 2^(nd) compression 27 wt % resilin, 60-100% glycerol, 100 0.7 3^(rd) compression 27 wt % resilin, propylene glycol, 64 3.9 1^(st) compression 27 wt % resilin, propylene glycol, 64 3.8 2^(nd) compression 27 wt % resilin, propylene glycol, 65 3.1 3^(rd) compression 27 wt % resilin, propylene glycol, 63 3.8 4^(th) compression 27 wt % resilin, propylene glycol, 61 3.9 5^(th) compression 27 wt % full length resilin, propylene glycol, 34 8.5 1^(st) compression 27 wt % full length resilin, propylene glycol, 39 7.7 2^(nd) compression 27 wt % full length resilin, propylene glycol, 37 7.7 3^(rd) compression

Example 8: Stiffness of Resilin Solids Vs. A Typical Midsole

Cross-linked silk solid compositions were prepared in water, 60-100% glycerol, 100% glycerol, propylene glycol (16 hour incubation), and propylene glycol (2 day incubation as described in Example 6. The stiffness of these materials was tested using a Shore 00 Durometer (AD-100-00) via ASTM: D-2240. A Dr. Scholl's Insole and a typical midsole were also tested for comparative purposes. The results are shown in Table 9.

TABLE 9 Stiffness of different resilin compositions vs. atypical midsole Solid Sample Description Hardness Scale Dr. Scholl's Insole 35-45 ◯◯ Typical midsole 55-63 ◯◯ Resilin - water-based 0-4 ◯◯ Resilin - 60% to 100% glycerol 10-16 ◯◯ Resilin - 100% glycerol 25-35 ◯◯ Resilin - 100% propylene glycol 30-40 ◯◯ Resilin - 100% propylene glycol - 2 d inc. 50 ◯◯ Resilin foam, 5% wt fumed silica - 60% to 10-30 ◯◯ 100% glycerol

Example 9: Elastic to Plastic Transition Measured by Zwick Compression Curves

Zwick compression data was obtained for 27 wt % resilin ethylene glycol solid (“A”), 27 wt % resilin propylene glycol solid (“C), coacervate resilin propylene glycol solid (“D”), 27 wt % resilin 60-100% glycerol solid (“G”), and 27 wt % resilin 100% glycerol solid (“H”). Each of these samples were prepared as described in Example 5.

Specifically, compression data for the above samples were tested using Zwick tensile tester with 5 kN load cell and 5 kN compression platens (Zwick/Roell Z5.0). Solid dimensions for each solid was a disc of 15 mm×5 mm. The materials were tested using a D695 Compressive Properties of Rigid Plastics compression program at a temperature of 22 C (ambient) and a humidity of 65% (ambient).

Force vs. strain curves as measured from the Zwick tensile tester for each solid are shown in FIG. 13. No elastic to plastic transition is visible for any of the resilin solids at up to 2 kN of applied compressive force. Instead, when the resilin solids broke under compressive stress it was via tearing and not elastic deformation.

Example 10: Analysis of Resilience and Compression of Propylene Glycol Resilin Solids

Rebound resilience and compressive stress at 25% strain was measured using a ASTM D7121 and ASTM D575 test for 27 wt % resilin solid exchanged into propylene glycol, prepared as in Example 5. The dimensions of the solid tested were a 1.125 inch diameter disc with a height of 0.8 inches. 3 different samples with these dimensions were submitted. The results are shown in Table 10.

TABLE 10 Data on resilience and compression of propylene glycol resilin solids Spec Ref Test Unit A B C ASTM D7121 Rebound resilience % 46.2 39.2 52.3 ASTM D575 Compressive stress at 25% psi 6.7 8.1 6.2 strain

The data shown in Table 10 can be compared to other rubbers or elastomers tested via the same ASTM test. The ASTM test number is sufficient to reproduce the results. This data and the durometer data are the only data that can be directly compared to other rubbers of elastomers.

ADDITIONAL CONSIDERATIONS

The foregoing description of the embodiments of the disclosure has been presented for the purpose of illustration; it is not intended to be exhaustive or to limit the claims to the precise forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the above disclosure.

The language used in the specification has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. It is therefore intended that the scope of the disclosure be limited not by this detailed description, but rather by any claims that issue on an application based hereon. Accordingly, the disclosure of the embodiments is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the claims.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

SEQUENCE LISTING SEQ ID NO: Species Sequence 1 Drosophila RPEPPVNSYLPPSDSYGAPGQSGAGGRPSDTYGAPGGGNGGRPSDSYGAPG sechellia QGQGQGQGQGGYGGKPSDSYGAPGGGNGNGGRPSSSYGAPGGGNGGRPSDT YGAPGGGNGGRPSDTYGAPGGGGNGNGGRPSSSYGAPGQGQGNGNGGRPSS SYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGNNGGRPSSSYGAP GGGNGGRPSDTYGAPGGGNGNGSGGRPSSSYGAPAQGQGGFGGRPSDSYGA PGQNQKPSDSYGAPGSGNGSAGRPSSSYGAPGSGPGGRPSDSYGPPASGSG AGGAGGSGPGGADYDNDEPAKYEFNYQVEDAPSGLSFGHSEMRDGDFTTGQ YNVLLPDGRKQIVEYEADQQGYRPQIRYEGDANDGSGPSGPSGPGGPGGQN LGADGYSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGG YSNVKPGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRP GGNGNGGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPG GRPGGNGQDSQDGQGYSSGRPGQGGRNGFGPGGQNGDNDGSGYRY 2 Drosophila RPEPPVNSYLPPSDSYGAPGQSGAGGRPSDTYGAPGGGNGGRPSDSYGAPG sechellia QGQGQGQGQGGYGGKPSDSYGAPGGGNGNGGRPSSSYGAPGGGNGGRPSDT YGAPGGGNGGRPSDTYGAPGGGGNGNGGRPSSSYGAPGQGQGNGNGGRPSS SYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGNNGGRPSSSYGAP GGGNGGRPSDTYGAPGGGNGNGSGGRPSSSYGAPAQGQGGFGGRPSDSYGA PGQNQKPSDSYGAPGSGNGSAGRPSSSYGAPGSGPGGRPSDSYGPPASG 3 Drosophila GKPSDSYGAPGGGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPS sechellia DTYGAPGGGGNGNGGRPSSSYGAPGQGQGNGNGGRPSSSYGAPGGGNGGRP SDTYGAPGGGNGGRPSDTYGAPGGGNNGGRPSSSYGAPGGGNGGRPSDTYG APGGGNGNGSGGRPSSSYGAPAQGQGGFGGRPSDSYGAPGQNQKPSDSYGA PGSGNGS 4 Acromyrmex FGENRGNGGKPSTSYGVPDSNGNNRGGFGNGGSEGRPSTSYGLPDASRNNG echinatior NGFGNVGNEDKPSTNYGIPANGNKVSGFGNVGSEGRPSTSYGVPGANGNQG FGSGGIGGRPSTSYGVPGVNGNNGGGFENVGRPSTSYGTPDARGNNGGSFR NGDIGGRPSTNYGIPGANGNHG 5 Aeshna APSRGGGHGGGSISSSYGAPSKGSGGFGGGSISSSYGAPSKGSVGGGVSSS YGAPAIGGGSFGGGSFGGGSFGGGSFGGGAPSSSYGAPSSSYSAPSSSYGA PSKGSGGFGSSGGFSSFSSAPSSSYGAPSASYSTPSSSYGAPSSGGFGAGG GFSSG 6 Aeshna EPPVGGSQSYLPPSSSYGAPSAGTGFGHGGGSPSQSYGAPSFGGGSVGGGS HFGGGSHSGGGGGGYPSQSYGAPSRPSGSSFQAFGGAPSSSYGAPSSQYGA PSGGGGSYAIQGGSFSSGGSRAPSQAYGAPSNNAGLSHQSQSFGGGLSSSY GAPSAGFGGQSHGGGYSQGGNGGGHGGSSGGGYSYQSFGGGNGGGHGGSRP SSSYGAPSSSYGAPSGGKGVSGGFVSQPSGSYGAPSQSYGAPSRGGGHGGG SISSSYGAPSKGSGGFGGGSISSSYGAPSKGSVGGGVSSSYGAPAIGGGSF GGGSFGGGSFGGGSFGGGAPSSSYGAPSSSYSAPSSSYGAPSKGSGGFGSS GGFSSFSSAPSSSYGAPSASYSTPSSSYGAPSSGGFGAGGGFSSGGYSGGG GGYSSGGSGGFGGHGGSGGAGGYSGGGGYSGGGSGGGQKYDSNGGYVYS 7 Haematobia AGGGNGGGGTGGTPSSSYGAPSNGGGSNGNGFGSPSSSYGAPGSGGSNGNG irritans GGRPSLSYGAPGSGGSNGNGGGRPSSSYGAPGAGGSNGNGGGRPSSSYGAP GAGGSNGNGGGRPSSSYGAPGAGGSNGNGGGRPSSSYGAPGAGGSNGNGGS RPSSTYGAP 8 Haematobia RPEPPVNSYLPPPLNNYGAPGAGGGSSDGSPLAPSDAYGAPDLGGGSGGSG irritans QGPSSSYGAPGLGGGNGGAPSSSYGAPGLGGGNGGSRRPSSSYGAPGAGGG NGGGGTGGTPSSSYGAPSNGGGSNGNGFGSPSSSYGAPGSGGSNGNGGGRP SLSYGAPGSGGSNGNGGGRPSSSYGAPGAGGSNGNGGGRPSSSYGAPGAGG SNGNGGGRPSSSYGAPGAGGSNGNGGGRPSSSYGAPGAGGSNGNGGSRPSS TYGAPGAGGSNGNGCGNKPSSSYGAPSAGSNGNGGSEQGSSGSPSDSYGPP ASGTGRGRNGGGGGAGGGRRGQPNQEYLPPNQGDNGNNGGSGGDDGYDYSQ SGDGGGQGGSGGSGNGGDDGSNIVEYEAGQEGYRPQTRYEGEANEGGQGSG GAGGSDGTDGYEYEQNGGDGGAGGSGGPGTGQDLGENGYSSGRPGGDNGGG GGYSNGNGQGDGGQDLGSNGYSSGAPNGQNGGRRNGGGQNNNGQGYSSGRP NGNGSGGRNGNGGRGNGGGYRNGNGNGGGNGNGSGSGSGNNGYNYDQQGSN GFGAGGQNGENDGSGYRYS 9 Ctenocephali ANGNGFEGASNGLSATYGAPNGGGFGGNGNGGAPSSSYGAPGAGNGGNGGG des felis  RPSSSYGAPGAGGSGNGFGGRPSSSYGAPGNGNGANGGRGGRPSSRYGAPG NGNGNGNGNGGRPSSSYGAPGSNGNGGRPSSSYGAPGSGNGFGGNGGRPSS SYGAPGANGNGNGGAIGQPSSSYGAPGQNGNGGGLSSTYGAPGAGNGGFGG NGGGLSSTYGAPGSGNGGFGGNGLSSTYGAPGSGNGGFGGNGGGLSSTYGA P 10 Ctenocephali PGGAGGAGGYPGGAGGAGGAGGYPGGSAGGAGGYPGGSGSGVGGYPGGSNG des felis GAGGYPGGSNGGAGGYPGGSNGGAGGYPGGSNGGAGGYPGGSNGNGGYSNG GSNGGGAGGYPGGSNGNGGYPGSGSNGGAGGYPGGSNGNGGYPG 11 Bombus FDGQNGIGGGDSGRNGLSNSYGVPGSNGGRNGNGRGNGFGGGQPSSSYGAP terrestris SNGLGGNGGSGAGRPSSSYGAPGGGNGFGGGQPSSSYGAPSNGLGGNGAGR PSSSYGAPGGGNGFGGGSNGAGKNGFGGAPSNSYGPPENGNGFGGGNGGGS PSGLYGPPGRNGGNGGNGGNGGNGGRPSSSYGTPERNGGRPSGLYGPP 12 Tribolium NGFGGGQNGGRLSSTYGPPGQGGNGFGGGQNGGRPSSTYGPPGQGGNGFGG costaneum GQNGGRPSSTYGPPGQGGNGFGGGQNGGRPSSTYGPPGQGGNGFGGGQNGG RPSSTYGPPGQGGNGFGGGQNGGRPSSTYGPPGQGGNGFGGGQNGGKPSST YGPPGQGGNGFGGGQNGGRPSSTYGPPGQG 13 Tribolium RAEPPVNSYLPPSQNGGPSSTYGPPGFQPGTPLGGGGNGGHPPSQGGNGGF costaneum GGRHPDSDQRPGTSYLPPGQNGGAGRPGVTYGPPGQGGGQNGGGPSSTYGP PGQGGNGFGGGQNGGRLSSTYGPPGQGGNGFGGGQNGGRPSSTYGPPGQGG NGFGGGQNGGRPSSTYGPPGQGGNGFGGGQNGGRPSSTYGPPGQGGNGFGG GQNGGRPSSTYGPPGQGGNGFGGGQNGGRPSSTYGPPGQGGNGFGGGQNGG KPSSTYGPPGQGGNGFGGGQNGGRPSSTYGPPGQGGNGNGGGHNGQRPGGS YLPPSQGGNGGYPSGGPGGYPSGGPGGNGGYGGEEESTEPAKYEFEYQVDD DEHNTHFGHQESRDGDKATGEYNVLLPDGRKQVVQYEADSEGYKPKISYEG GNGNGGYPSGGPGGAGNGGYPSGGPQGGNGGYPSGGPQGGNGGYPSGGPQG GNGGYPSGGPQGGNGGYPSGGPQGGNGGYPSGGPQGGNGGYPSGGPQGGNG GYPSGGPQGGNGGYTSGGPQGGNGGYPSGGPQGGNGGSGPY 14 Tribolium QLTKRDAPLSGGYPSGGPANSYLPPGGASQPSGNYGAPSGGFGGKSGGFGG costaneum SGGFGGAPSQSYGAPSGGFGGSSSFGKSGGFGGAPSQSYGAPSGGFGGSSS FGKSSGGFGGAPSQSYGAPSGGFGGSSSFGKSGGFGGAPSQSYGAPSGGFG GSSSFGKSGGFGGAPSQSYGAPSGGFGGKSSSFSSAPSQSYGAPSGGFGGK SGGFGGAPSQSYGAPSGGFGGKSGGFGGAPSQSYGAPSGGFGGSSSFGKSG GFGGAPSQSYGAPSGGFGGSSSFGKSSGFGHGSGAPSQSYGAPSRSQPQSN YLPPSTSYGTPVSSAKSSGSFGGAPSQSYGAPSQSHAPSQSYGAPSRSFSQ APSQSYGAPSQGHAPAPQQSYSAPSQSYGAPSGGFGGGHGGFGGQGQGFGG GRSQPSQSYGAPAPSQSYGAPSAGGQQYASNGGYSY 15 Apis  RSEPPVNSYLPPSGNGNGGGGGGSSNVYGPPGFDGQNGIGEGDNGRNGISN mellifera SYGVPTGGNGYNGDSSGNGRPGTNGGRNGNGNGRGNGYGGGQPSNSYGPPS NGHGGNGAGRPSSSYGAPGGGNGFAGGSNGKNGFGGGPSSSYGPPENGNGF NGGNGGPSGLYGPPGRNGGNGGNGGNGGRPSGSYGTPERNGGRLGGLYGAP GRNGNNGGNGYPSGGLNGGNGGYPSGGPGNGGANGGYPSGGSNGDNGGYPS GGPNGNGNGNGGYGQDENNEPAKYEFSYEVKDEQSGADYGHTESRDGDRAQ GEFNVLLPDGRKQIVEYEADQDGFKPQIRYEGEANSQGYGSGGPGGNGGDN GYPSGGPGGNGYSSGRPNGGSDFSDGGYPSTRPGGENGGYRNGNNGGNGNG GYPSGNGGDAAANGGYQY 16 Apis  DAPISGSYLPPSTSYGTPNLGGGGPSSTYGAPSGGGGGRPSSSYGAPSSTY mellifera GAPSSTYGAPSNGGGRPSSTYGAPSNGGGRPSSSYGAPSSSYGAPSSTYGA PSNGGGRPSSSYGAPSFGGGGGFGGGNGLSTSYGAPSRGGGGGGGSISSSY GAPTGGGGGGPSTTYGAPNGGGNGYSRPSSTYGTPSTGGGSFGGSGGYSGG GGGYSGGGNGYSGGGGGGYSGGNGGGYSGGGNGGGYSGGNGGGYSGGGGGG YSGGGGGGYSGGGNGYSGGGGGGYSGGNGGYSGGNGGYSGGGGGYSGGGGG GQSYASNGGYQY 17 Nasonia RPEPPVNSYLPPGQGGGFGGGRPSGASPSDQYGPPDFQGAGGRGGQAAGGN vitripennis FGGGGNGFGGAPSSSYGPPGFGSNEPNKFSGAGGGGAGRPQDSYGPPAGGN GFAGSAGAGNSGRPGGAAAGGRPSDSYGPPQGGGSGFGGGNAGRPSDSYGP PSAGGGGFGGGSPGGGFGGGSPGGGFGGGNQGAPQSSYGPPASGFGGQGGA GQGRPSDSYGPPGGGSGGRPSQGGNGFGGGNAGRPSDSYGPPAAGGGGFGG NAGGNGGGNGFGGGRPSGSPGGFGGQGGGGRPSDSYLPPSGGSGFGGGNGR QPGGFGQQGGNGAGQQNGGGGAGRPSSSYGPPSNGNGGGFSGQNGGRGSPS SGGGFGGAGGSPSSSYGPPAGGSGFGNNGGAGGRPSSSYGPPSSGGNGFGS GGQGGQGGQGGQGGRPSSSYGPPSNGNGGFGGGNGGRPSSNGYPQGQGNGN GGFGGQGGNGGRPSSSYGPPGGDSGYPSGGPSGNFGGSNAGGGGGGFGGQV QDSYGPPPSGAVNGNGNGYSSGGPGGNGLDEGNDEPAKYEFSYEVKDDQSD GRKQIVEYEADQDGFKPQIRYEGEANTGAGGAGGYPSGGGGDSGYPSGPSG AGGNAGYPSGGGGGAGGFGGNGGGSNGYPSGGPSGGQGQFGGQQGGNGGYP SGPQGGSGFGGGSQGSGSGGYPSGGPGGNGGNNNFGGGNAGYPSGGPSGGN GFNQGGQNQGGSGGGYPSGSGGDAAANGGYQYS 18 Alasonia RAEAPISGNYLPPSTSYGTPNLGGGGGGGGGFGGGAPSSSYGAPSSGGGFG vitripennis GSFGGGAPSSSYGAPSTGGSFGGGAPSSSYGAPSSGGSFGGSFGGGAPSSS YGAPSFGGNAPSSSYGAPSAGGSFGGGAPSNSYGPPSSSYGAPSAGGSFGG SSGGSFGGSFGGGAPSSSYGAPAPSRPSSNYGAPSRPSSNYGAPSSGGSGF GGGSGFGGGRPSSSYGAPSSGSFGGGFGGGAPSSSYGAPAPSRPSSNYGAP APSRPSSNYGAPAPSRPSSSYGAPSRPSSNYGAPSRPSSNYGAPSSGGSGF GGGSGFGGGRPSSSYGAPSSGSFGGGFGGGAPSSSYGAPAPSRPSSNYGPP SSSYGAPSSGGSGGFGGGAPSSSYGAPSFGGSSNAVSRPSSSYGAPSSGGG QSYASNGGYQY 19 Pediculus EPPVKTSYLPPSASRSLNSQYGAPAFTDSNELVAPSPNSNFHDSYNQQQQS hurnanus FDLSNGLSVPSAAGRLSNTYGVPSAQGANVPSFDSSDSIAVDAAGRSGNSF corporis SSHVPSSTYGAPGNGFGGGSRSSQSGAPSSVYGPPQARNNNFGNGAAPSSV YGPPQARNNNFGNGGAPSQVYGPPKARNNNFGNGAAPSSVYGPPQARNNNF GNGAAPSSVYGPPQARNNNFANSAAPSQVYGPPQARNNNFGNGAAPSSVYG PPQSSSFSSPSGRSGQLPSATYGAPFERNGFGSQGSSGFQGYEPSKRSQTT EDPFAEPAKYEYDYKVQASDETGTEFGHKESRENESARGAYHVLLPDGRMQ IVQYEADETGYRPQIRYEDTGYPSAASSRSNNGFNGYQY 20 Anopheles KREAPLPPSGSYLPPSGGAGGYPAAQTPSSSYGAPTGGAGSWGGNGGNGGR gambiae  GHSNGGGSSFGGSAPSAPSQSYGAPSFGGQSSGGFGGHSSGGFGGHSSGGH str. GGNGNGNGNGYSSGRPSSQYGPPQQQQQQQSFRPPSTSYGVPAAPSQSYGA PEST PAQQHSNGGNGGYSSGRPSTQYGAPAQSNGNGFGNGRPSSSYGAPARPSTQ YGAPSAGNGNGYAGNGNGRSYSNGNGNGHGNGHSNGNGNNGYSRGPARQPS QQYGPPAQAPSSQYGAPAQTPSSQYGAPAQTPSSQYGAPAQTPSSQYGAPA QTPSSQYGAPAPSPPSQQYGAPAPSRPSQQYGAPAQTPSSQYGAPAQTPSS QYGAPAQTPSSQYGAPAQTPSSQYGAPAQQPSSQYGAPAPSRPSQQYGAPA QQPSAQYGAPAQTPSSQYGAPAPSPPSQQYGAPAQAPSSQYGAPAPSSQYG APAQQPSSQYGAPAQTPSSQYGAPSFGPTGGASFSSGNGNVGGSYQVSSTG NGFSQASFSASSFSPNGRTSLSAGGFSSGAPSAQSAGGYSSGGPSQVPATL PQSYSSNGGYNY 21 Glossina RPEPPVNTYLPPSAGGGSGGGSPLAPSDTYGAPGVNGGGGGGGGPSSTYGA morsitans PGSGGGNGNGGGGFGKPSSTYGAPGLGGGGNGGGRPSETYGAPSGGGGNGF GKPSSTYGAPNGGGGNGGPGRPSSTYGAPGSGGGNGGSGRPSSTYGAPGLG GGNGGSGRPSSMYGAPGLGGGNGGSGRPSSTYGAPGSGGGNGGSGRPSSTY GAPGSGGGNGGSGRPSSTYGAPGNGNGGNGFGRPSSTYGAPGSGGSNGNGK PSSTYGAPGSGGGGGRPSDSYGPPASGNGGRNGNGNGQSQEYLPPGQSGSG GGGGYGGGSGSGGSGGGGGGGYGGDQDNNVVEYEADQEGYRPQTRYEGDGS QGGFGGDGDGYSYEQNGVGGDGGGAGGAGGYSNGQNLGANGYSSGRPNGGN GGGRRGGGGGGGGSGGGQNLGSNGYSSGAPNGFGGGNGQGYSGGRSNGNGG GGGGRNGGRYRNGGGGGGGRNGGGSNGYNYDQPGSNGFGRGGGNGENDGSG YHY 22 Atto RSEPPVNSYLHPGSDTSGTNGGRTDLSTQYGAPDFNNRGNGNSGATSFGGS cepholotes GAGNGPSKLYDVPIRGNTGGNGLGQFRGNGFESGQPSSSYGAPKGGFGENR GNRGRPSTSYGVPDSNRNNRGGFGNGGSEARPSTSYGVPGANGNQGGFGSG SIGGRPSTSYGVPGANGNNGDSFRNGDIGGRPSTNYGAPGANGNHGGGNGG NGRPSNNYGVPGANGNTNGKGRLNGNSGGGPSNNYGSPNGFGKGLSTSYGS PNRGGNDNHYPSRGSFINGGINGYSSGSPNGNAGNFGHGDESFGRGGGEGE NTGEGYNANAQEESTEPAKYEFSYKVKDQQTGSDYSHTETRDGDHAQGEFN VLLPDGRKQIVEYEADQDGFKPQTRYEGEANADGGYGSGLNDNNDGYSSGR PDSESGGFANSGFNGGSSNGGYPNGGPGERKLGGFNNGGSSGYQSGRSAGQ SFGRDNAGDLNNDIGGYFSNSPNNIGDSDNANVGSNRQNDGNSGYQY 23 Anopheles KREAPLPPSGSYLPPSGGGGGGGGYPAAQTPSSSYGAPAGGAGGWGGNGNG clarlingi NGNGNGGRGGYSNGGGHSGSAPSQSYGAPSAPSQSYGAPSQSYGAPAAAPS QSYGAPSFGGNGGGASHGSGGFTGGHGGNGNGNGYSSGRPSSQYGPPQQQQ QPQQQSFRPPSTSYGVPAAPSSSYGAPSANGFSNGGRPSSQYGAPAPQSNG NEFGAPRPSSSYGAPSRPSTQYGAPSNGNGNGYAGHGNGNGHGNGNGHSNG NGNGYNRGPARQPSSQYGPPSQGPPSSQYGPPSQYGPPSSGTSFIAYGPPS QGPPSSQYGAPAPSRPSSQYGAPAQTPSSQYGAPAQTPSSQYGPPRQSSPQ FGAPAPRPPSSQYGAPAQAPSSQYGAPAQTPSSQYGAPAQAPSSQYGAPAP SRPSSQYGVPAQAPSSQYGAPAQAPSSQYGAPAQTPSSQYGAPSFGSTGGS SFGGNGGVGGSYQTASSGNGFSQASFSASSFSSNGRSSQSAGGYSSGGPSQ VPATIPQQYSSGGGSYSSGGHSQVPATLPQQYSSNGGYNY 24 Acromyrmex RSEPPVNSYLPPGPGTSGANGGQTDLSIQYRASDFNNRGNVNGNSGATSFG echinatior GPGASNGPSKLYDVPIGGNAGGNGLGQFRGNGFEGGQPSSSYGAPNGGFGE NRGNGGKPSTSYGVPDSNGNNRGGFGNGGSEGRPSTSYGLPDASRNNGNGF GNVGNEDKPSTNYGIPANGNKVSGFGNVGSEGRPSTSYGVPGANGNQGFGS GGIGGRPSTSYGVPGVNGNNGGGFENVGRPSTSYGTPDARGNNGGSFRNGD IGGRPSTNYGIPGANGNHGGGNGGNGRPSSNYGVPGGNGNTNGKGRFNGNS GGRPSNSYGSPNGFGKGLSTSYSPSNRDGNGNHYPSGDSNRGSFVNGGING YPSGSPNGNAGNFRHGDESFGRGGEGGGRSTGEGYNANAQEESTEPAKYEF SYKVKDQQTGSDYSHTETRDGDHAQGEFNVLLPDGRKQIVEYEADQDGFKP QIRYEGEANADGEYDSGGLNDNNDGYSSGRPGSESGGFANNSGFNGGSSNG GYPSGGSGEGKLGFNSGGNSGYQSGRPAGQSFGRDNAGDLSNDIGGFSNSP NNIGGDNANVGSNRQNGGNSGYQY 25 Acyrthosiphon ESPYGGGSSNSNGNGRNGGYGGKGQYGGGNGGGVGSSSASPFFSGANQYGS pisum QSGLSGAANNRYPSFGSKFGGNKGSYGGSSSRNNGRYGSGSASGYGSGSSG GLGSTGRSTGGYGGGSSGSYGSGSSGSLGSSTGSNGIYGAGSSGGFGSGSS GSYGGGSSGGFGSGSSGSYGGGSSGGFGSGSSGSYGGGSSGGFGSGSSGSY GGGSSGGFGSGSSGNYGSGSSGSYGSGGGGLGGASSGNNDGYGAGGSGSYD QLGGANGNGLGGSGNDPLSEPANYEFSYEVNAPESGAIFGHKESRQGEEAT GVYHVLLPDGRTQIVEYEADEDGYKPKITYTDPVGGYAGDRQSGNSYGGNG GFGGSGSLGGSGGNLGGLYNGGGSSNNGAGYGGSSSSLGSRYGGSGGSSGS GVGGGYGGSGSSSGGIGSSYGGSGSLSGGLGGGYGGSGSSSGGLGGGYGGS GGSSGGGFGGLGGSGGSSGSGYGGSGSSSGGLGNSYGGSGSSNGGLGGGYS GSGGSSGGLGGGYGASSGSSGSGLGGGYGGSGSSSGGLGSGYGGLGSSSGG LGGGYGGSGSSSGGLGGGYGGSGSSNGGIGGGYGGSSGSSGGLGGGYGGSG SSSGGLGGGYGGSGGSNSGLGSSYGGSGSTNGGLGGGYGGLGSSSGGLGGG YGGSGGSNGGIGGGYGGSSGSGGSQGSAYGGSGSSSGSQGGGYGGSGSSSG GLGGGYGSSSGSSSGLGGSYGSNRNGLGSGSSYS 26 Drosophila RPEPPVNSYLPPSPGDSYGAPGQGQGQGQGGFGGKPSDSYGAPGAGNGNGN virilis GRPSSSYGAPGQGQGQGGFGGKPSDSYGAPGAGNGNGNGRPSSSYGAPGQG QGQGGFGGRPSSSYGAPGQGQGGFGGKPSDTYGAPGAGNGNGRPSSSYGAP GQGQGGIGGKPSDSYGAPGAGNGNGNGRPSSSYGAPGQGQGGFGGKPSDTY GAPGAGNGNGRPSSSYGAPGQGQGGFGGKPSDTYGAPGAGNGNGNGRPSSS YGAPGQGQGGFGGKPSDTYGAPGAGNGNGRPSSSYGAPGQGQGQGGFGGKP SDSYGPPASGAGAGGAGGPGAGGGGDYDNDEPAKYEFNYQVEDAPSGLSFG HSEMRDGDFTTGQYNVLLPDGRKQIVEYEADQQGYRPQVRYEGDANGNGGP GGAGGPGGQDLGQNGYSSGRPGGQDLGQGGYSNGRPGGQDLGQNGYSGGRP GGQDLGQNGYSGGRPGGQDLGQNGYSGGRPGGQDLGQNGYSGGRPGGQDLG QNGYSGGRPGGQDLGQNGYSGGRPGGNGGSDGGRVIIGGRVIGQDGGDGQG YSSGRPNGQDGGFGQDNTDGRGYSSGKPGQGRNGNGNSFGPGGQNGDNDGS GYRY 27 Drosophila RPEPPVNSYLPPSDSYGAPGQSGPGGRPSDSYGAPGGGNGGRPSDSYGAPG erecta LGQGQGQGQGQGGFGGKPSDSYGAPGAGNGNGGRPSSSYGAPGAGNGGRPS DTYGAPGGGSGGRPSDTYGAPGGGNGNGNGGRPSSSYGAPGQGQGNGNSGR PSSSYGAPGAGNGGRPSDTYGAPGGGNGGRPSSSYGAPGAGNGGNGGRPSD TYGAPGGGNGNGNGNGNGSGGRPSSSYGAPGQGQGGFGGRPSDSYGAPGQN QKPSDSYGAPGSGSGSGNGNGGRPSSSYGAPGSGPGGRPSDSYGPPASGSG AGGAGGSGPGGADYDNDIVEYEADQQGYRPQIRYEGDANDGSGPSGPGGQN LGADGYSSGRPGNGNGNGNGGYSGGRPGGQDLGPSGYSGGRPGGQDLGAGG YSNGRPGGQDLGPSGYSGGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNGRP GGNGNGNGGADGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGR PGGRPGANGQDNQDGQGYSSGRSGKGGRNSFGPGGQNGDNDGSGYRY 28 Lutzomyia RPEPPANTYLPPSSSYAAPGQQGGSGFGGGGGSGGSGGFGQPGAFGRPSSS longipalpis YGPPSQGGAGGGFGSDSQFGGGFGGGAGGFGSGGSGAPGASQRPSSSYGPP GQTGGGGFGAQGAPGSSFGPGGGFGGGSPGQAGSPGFQRPSSSYGPPGQSP GGGFSQQGGAPGASQRPSSTYGAPGQGAGGFGQGGSGGFGGTGGSVAIGGR PSSSYGAPGQGSSGGFGGGSGGFGSQAPSTSYGAPGQGSPGGGFGSQGGPG GQPGSPGFGGSQRPSSSYGPPGQGGAPGQGGSPGFGASSRSGGAGGFGASQ QPSSSYGPPGQGAGSGFQGTGGGFGGPGQRPGFGGSQTPATSYGAPGQAGG ASGGFGGAGAQRPSSSYGPPGQASGFGGGSSGGGFGGGSSGGFGGNQGGFG GNQGGFGGSQTPSSSYGAPSFGSGGSPGAAGGAGGFGQGGVGGSGQPGGFG GGDQGYPPRGGPGGFGPGSGGSGAGGPIAGGSGSGYPGGSDSGSNEPAKYD FSYQVDDPASGTSFGHSEQRDGDYTSGQYNVLLPDGRKQIVEYEADLGGYR PQIKYEGGSSGGAGGYPSGGPGSQGGAGGYPSGGPGGPGSPGGAGGYQSGA AGGAGGYPSGGPGGPGAGGYPSGGPGGPGSQAGGFSGGFGGGSDGAFGGAG GFSQGGAGGGDAGYPRGGPGGFGGAGSPGFGGSGSPGFGGSGSPGAQGSSG FGGTGGGFGGGADGYPRGGPGAGQSGFQDGRGATGGAGQPGGRGSFGRPGS ARGGSSSNGYANGGAEGYPRDNPQNRGSGYS 29 Rhodnius KRDDPLRRFLAPLVGGGNGSGGGGGGYNYNKPANGLSLPGGGGALPPATSY prolixus GVPDRPAPVPSSPPSSSYGAPQPSPNYGAPSSSYGAPSQQPSRSYGAPSQG PSTSYSQRPSSSYGAPAPQTPSSSYGAPAQQPSGSYGAPSGGGGSSGYTGG AQRPSGSYGAPSQGGPSGNYGPPSQQPSSNYGAPSQTPSSNYGAPAQRPST SYGAPSQPPSSSYGSPPQRASGYPSSSSGPSNGYSPPAQRPSSSYGPPSQQ PASSYGAPSQTPSSNYGPPAPIPSSNYGAPSQPPSKPSAPSSSYGTPSQTP STSYGAPSQAPSSSYGAPSRPSPPSSSYGAPSQGPSSSYGPPSRPSQPSSP SSGYGAPSQGPSSSYGAPSRPSSPSSSYGAPPSSSYGAPSRPSPPSSSYGA PSQGPSSSYGPPSRPSQPSSPSSGYGAPSQGPSSSYGAPSRPSSPSSSYGA PPSSSYGAPSRPSPPSSSYGAPSQGPSSSYGPPSRPSQPSSPSSSYGAPSQ GPSSSYGAPSRPSPPSSSYGAPSQGPSSSYGPPSRPSQPSSTYGVPSGGRP STPSSSYGAPPQALSSTYGAPSGRPGAPSQKPSSSYGAPSLGGNASRGPKS SPPSSSYGAPSVGTSVSSYAPSQGGAGGFQSSRPSSSYGAPSTGPSSTYGP PSQPPSSSYGVPSQPPSSNYGVPSQGVSGSVGSSSPSSSYGAPSQIPSSSY GAPSQSSIGGFGSSRPSSSYGAPPQAPSSSYSAPLRAPSTSYGAPSGGSGS NFGSKPSTNYGAPSQPPSTNYGPPSQPPSSSYGTPSRAPSPTYSTPQSSGT SFGSRPSSSYGVPSQPTTNYGAPSQTPSSNYGAPPASSAPSSTYGRPSQSP SSSYGAPSPSSSSSSYESPSQPPSSSYGAPSQGPSSSYGAPSRPSSTYGAP SPSSPSTNYGAPAPSSNYGTPAQDLTGSYAAPSQPPSAGYGAPSGQPSSGG KQNFQVKNPFAGQTHQVYPAVSSISFGLPSQSFNTAIQGQEPSQSYGAPTA SSPSSSYGAPTGTGSSQPGQSYASNGGYSYS 30 Rhodnius QPPFNHYLPAARGSGSNSAQYTAPSSKFGTSTGQYGQPPSEVPRGLQQGSY prolixus AEDVHSSRSVNPSSQNGIPSGHFSSLSSNYGAPSSDYSRSFLRYGTLSNKY GVPNSALGSLSSRNNKTPATQLSYQPSSHYDSRSTSEDQFISSRVSDSQYG ASSVRRFLPSSQYSTPSSQYGTPSSQYGTPSSQYGTPSSQYGTPSSQYGTP SSQYGTPSSQYGTPSSQYGTPSSQYGTPSSQYGTPSSPPSQYGGPYSMRTS APNSQYGTPSSFRTSPSSQFGSSSAHSSSLSKFRSVPSSPYGTLSAIRSTH SSQYGTPSSFSDSTSSSHNGLPSHYPGSGFSGSSVNDQKSYTGNVFGQSHS RVANGDQHARSYTLAGGNEISEPAKYDFNYDVSDGEQGVEFGQEESRDGEE TNGSYHVLLPDGRRQRVQYTAGQYGYKPTISYENTGTLTTGRQQFSNGFYN VQQSGSESQEHLGRSTGQNSYGGSNGYESGVGYQSGVGRRSRPAGSY 31 Solenopsis RSEPPINSYLPPRAGSSGANGGRTDLTTQYGAPDFNNGGGATSFSGNGAGD invicto GPSKLYDVPVRGNAGGNGLGRGNGFGGGQPSSSYGAPNGGSNENRGNGGRP STSYGVPGANGNNGGGFGNGGDKGRPSTSYGVPDASGSSQGSFGNVGNGGR PSTNYGVPGANGNGGGFGNAANEGKPSTSYGVPGANGNSQGGFGNGGRPST GYGVPGANGNNGGGFGGRPSTSYGAPGANGNHRGGNGGNASPSTNYGVPGG NNGNTNGKGRFNGGNSGGGPSNNYGVPNENAFGGGLSTSYGPPSRGGNGNS GYPSGGSNGGSFVNNGANGYPSGGPNGNAGNFGDGRGGKGGGSSGEGYNDN AQEGSTEPAKYEFSYKVKDQQTGSEYSHTETRDGDRAQGEFNVLLPDGRKQ IVEYEADQDGFKPQIRYEGEANAGGGYSSGGSNDNNDGYSSGRPGSEAGGF ANNSGFNGSGTNGGRSSGGPGDGNPGGFNSGGGGGYQSGRPAGQSFGRDND GGLSGDIGGYFANSPSNNIGGSDSANVGSNRQNGGNGGYQY 32 Culex KREAPLPGGSYLPPSNGGGAGGYPAAGPPSGSYGPPSNGNGNGNGAGGYPS quinque- APSQQYGAPAGGAPSQQYGAPSNGNGGAGGYPSAPSQQYGAPNGNGNGGFG fasciatus GRPQAPSQQYGAPSNGNGGARPSQQYGAPNGGNGNGRPQTPSSQYGAPSGG APSSQYGAPSGGAPSQQYGAPNGGNGNGRPQTPSSQYGAPSGGAPSQQYGA PNGGNGNGRPQTPSSQYGAPSGGAPSSQYGAPSGGAPSSQYGAPAGGAPSS QYGAPAGGAPSSQYGAPAGGAPSSQYGAPAGGAPSSQYGAPAGGAPSSQYG APSSQYGAPAGGAPSSQYGAPAGGAPSSQYGAPSGGAPSSQYGAPSGGAPS SQYGAPAGGAPSSQYGAPSGGAPSS 33 Bactrocera RPEPPVNSYLPPSANGNGNGGGRPSSQYGAPGLGSNSNGNGNGNGGGRPSS cucurbitae QYGVPGLGGNGNGNGNGGGGGRPSSSYGAPGLGGNGNGNGNGGGRPSSQYG VPGLGGNGNGNGNGNGGGRPSSTYGAPGLRGNGNGNGNGNGRPSSTYGAPG LGGNGNGNGNGNGRPSSTYGAPGLGGNGNGNGNGNGRPSSTYGAPGLNGNG LGGGQKPSDSYGPPASGNGNGYSNGGNGNGNGGGRPGQEYLPPGRNGNGNG NGGRGNGNGGGANGYDYSQGGSDSGESGIVDYEADQGGYRPQIRYEGEANN GAGGLGGGAGGANGYDYEQNGNGLGGGNGYSNGQDLGSNGYSSGRPNGNGN GNGNGNGNGYSGRNGKGRNGNGGGQGLGRNGYSDGRPSGQDLGDNGYASGR PGGNGNGNGGNGNGYSNGNGYSNGNGNGTGNGGGQYNGNGNGYSDGRPGGQ DNLDGQGYSSGRPNGFGPGGQNGDNDGNGYRY 34 Trichogramma RPEPPVNSYLPPGQGGQGGFGGSGGRPGGGSPSNQYGPPNFQNGGGQNGGS pretiosum GFGGNGNGNSFGPPSNSYGPPEFGSPGAGSFGGGRPQDTYGPPSNGNGNGN GFGGNGNGGGRPSSRPSDSYGPPSSGNGFGGGNSGRPSESYGPPQNGGGSG NGNQGGGNGFGNGGGRGGQGKPSDSYGPPNSGNRPGSSNGGGQQQNGFGGG NGGRPSNTYGPPGGGNGGGRPGGSSGGFGGQNGGRPSDSYGPPSNGNGNGG RPSNNYGPPNSGGGNGNGFGGSNGKPSNSYGPPSNGNGGGFGGSNGRPSNS YGPPSGGNGGGFGGSSAVGRPGNSGSPSSSGSGFGGNGGASRPSSSYGPPS NGGGFGNGGGSNGRPSSSYGPPNSGSNGGGFGGQNGNGRQNGNNGQGGFGG QPSSSYGPPSNGNGFGGGGGSNGYPQNSQGGNGNGFGQGSGGRPSSSYGPP SNGGGGGDNGYSSGGPGGFGGQPQDSYGPPPSGAVDGNNGFSSGGSSGDNN GYSSGGPGGNGFEDGNDEPAKYEFSYEVKDEQSGSSFGHTEMRDGDRAQGE FNVLLPDGRKQIVEYEADQDGFKPQIRYEGEANTGGAGGYPSGGPGGQGGN GNGGYPSGGPSNGGFGGQNGGGNGGYPSGGPSGGGFGGQNGGSGGYPSGGP SGGGFGGQGGFGGQNSGGNGGYSSGGPASGGFGGQNGGNGGYPSGGPSGGG FGGQGGFGGQNSGGNGGYPSGGPSSGGFGGQNGGGGGNYPAGSGGDAEANG GYQYS 35 Drosophila QSGAGGRPSDTYGAPGGGNGGRPSDSYGAPGQGQGQGQGQGGYGGKPSDSY sechellia GAPGGGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPG GGGNGNGGRPSSSYGAPGQGQGNGNGGRPSSSYGAPGGGNGGRPSDTYGAP GGGNGGRPSDTYGAPGGGNNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNG NGSGGRPSSSYGAPAQGQGGFGGRPSDSYGAPGQNQKPSDSYGAPGSGNGS AGRPSSSYGAPGSGPGGRPSDSYGP 36 Drosophila QSGAGGRPSDTYGAPGGGNGGRPSDSYGAPGQGQGQGQGQGGYGGKPSDSY sechellia GAPGGGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPG GGGNGNGGRPSSSYGAPGQGQGNGNGGRPSSSYGAPGGGNGGRPSDTYGAP GGGNGGRPSDTYGAPGGGNNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNG NGSGGRPSSSYGAPAQGQGGFGGRPSDSYGAPGQNQKPSDSYGAPGSGNGS AGRPSSSYGAPGSGPGGRPSDSYGP 37 Drosophila QSGAGGRPSDTYGAPGGGNGGRPSDSYGAPGQGQGQGQGQGGYGGKPSDSY sechellia GAPGGGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPG GGGNGNGGRPSSSYGAPGQGQGNGNGGRPSSSYGAPGGGNGGRPSDTYGAP GGGNGGRPSDTYGAPGGGNNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNG NGSGGRPSSSYGAPAQGQGGFGGRPSDSYGAPGQNQKPSDSYGAPGSGNGS AGRPSSSYGAPGSGPGGRPSDSYGP 38 Drosophila QSGAGGRPSDTYGAPGGGNGGRPSDSYGAPGQGQGQGQGQGGYGGKPSDSY sechellia GAPGGGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPG GGGNGNGGRPSSSYGAPGQGQGNGNGGRPSSSYGAPGGGNGGRPSDTYGAP GGGNGGRPSDTYGAPGGGNNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNG NGSGGRPSSSYGAPAQGQGGFGGRPSDSYGAPGQNQKPSDSYGAPGSGNGS AGRPSSSYGAPGSGPGGRPSDSYGP 39 Drosophila YSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNVK sechellia PGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRPGGNGN GGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPGGRPGG NGQDSQDGQ 40 Drosophila YSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNVK sechellia PGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRPGGNGN GGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPGGRPGG NGQDSQDGQ 41 Drosophila YSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNVK sechellia PGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRPGGNGN GGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPGGRPGG NGQDSQDGQ 42 Drosophila YSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNVK sechellia PGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRPGGNGN GGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPGGRPGG NGQDSQDGQ 43 Drosophila YSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNVK sechellia PGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRPGGNGN GGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPGGRPGG NGQDSQDGQ 44 Drosophila YSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNVK sechellia PGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRPGGNGN GGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPGGRPGG NGQDSQDGQ 45 3XFLAG GDYKDDDDKDYKDDDDKDYKDDDDK 46 Alpha mating MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVA factor VLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR precursor protein sequence 47 EA repeat EAEA 48 Linker SG 49 full-length MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVA Drosophila VLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR EAEAgrpeppynsylpp sechellia sdsygapgqsgaggrpsdtygapgggnggrpsdsygapgqgqgqgqgqggy resilin ggkpsdsygapgggngnggrpsssygapgggnggrpsdtygapgggnggrp sequence sdtygapggggngnggrpsssygapgqgqgngnggrpsssygapgggnggr (Ds_ACB) psdtygapgggnggrpsdtygapgggnnggrpsssygapgggnggrpsdty gapgggngngsggrpsssygapaqgqggfggrpsdsygapgqnqkpsdsyg apgsgngsagrpsssygapgsgpggrpsdsygppasgsgaggaggsgpgga dydndepakyefnyqvedapsglsfghsemrdgdfttgqynvllpdgrkqi veyeadqqgyrpqiryegdandgsgpsgpsgpggpggqnlgadgyssgrpg ngngngnggyssgrpggqdlgpsgysggrpggqdlgaggysnvkpggqdlg pggysggrpggqdlgrdgysggrpggqdlgagaysngrpggngnggsdggr viiggrviggqdggdqgysggrpggqdlgrdgyssgrpggrpggngqdsqd gqgyssgrpgqggrngfgpggqngdndgsgyry

DYKDDDDKDYKDDDDK DYKDDDDK 

1. A method of cross-linking recombinant resilin, comprising: providing a composition comprising purified recombinant resilin; placing said recombinant resilin in a cross-linking solution comprising ammonium persulfate; and incubating said recombinant resilin in said cross-linking solution at a temperature of at least 60° C., thereby generating a cross-linked recombinant resilin solid composition.
 2. The method of claim 1, wherein said incubation is performed for at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, or at least 2 hours.
 3. The method of claim 1, wherein said recombinant resilin in said cross-linking solution is incubated at a temperature of from 60° C. to 85° C., from 70° C. to 85° C., or from 75° C. to 85° C.
 4. (canceled)
 5. The method of claim 1, wherein said cross-linking solution does not comprise a photocatalyst or a cross-linking enzyme.
 6. The method of claim 1, wherein said cross-linked recombinant resilin solid composition is stable at room temperature for more than 5 days, more than 10 days, more than 20 days, or more than 40 days.
 7. The method of claim 1, wherein said purified recombinant resilin is prepared by recombinantly expressing a gene encoding said recombinant resilin in a modified organism in a culture, and purifying expressed recombinant resilin from said culture. 8.-10. (canceled)
 11. A recombinant resilin composition comprising a cross-linked recombinant resilin in a polar nonaqueous solvent. 12.-25. (canceled)
 26. The method of claim 1, wherein said cross-linking solution is an aqueous solvent; and wherein said method further comprises exchanging said aqueous solvent with a polar nonaqueous solvent.
 27. The method of claim 26, wherein said polar nonaqueous solvent is a protic solvent.
 28. The method of claim 27, wherein said protic solvent is selected from the group consisting of: glycerol, propylene glycol, and ethylene glycol.
 29. The method of claim 26, wherein said polar nonaqueous solvent is an aprotic solvent.
 30. The method of claim 29, wherein said aprotic solvent is DMSO.
 31. The method of claim 26, wherein said solvent exchange is performed for at least 8 hours, at least 16 hours, at least 24 hours, or at least 48 hours.
 32. The method of claim 26, wherein said solvent exchange is performed at about 60° C.
 33. The method of claim 26, wherein said cross-linked recombinant resilin solid composition comprises at least 20%, at least 50%, at least 70%, or at least 80% full length resilin as a portion of all resilin as measured by size exclusion chromatography.
 34. (canceled)
 35. (canceled)
 36. The method of claim 26, wherein exchanging said solvent changes the elastic modulus, the resilience, the hardness, the maximum elastic compressive load, or the material lifetime of the cross-linked recombinant resilin solid composition.
 37. (canceled)
 38. A method of preparing a recombinant resilin foam, comprising: providing a cross-linked recombinant resilin solid composition in an aqueous solvent; exchanging said aqueous solvent with a polar nonaqueous solvent; and introducing one or more bubbles to the cross-linked recombinant resilin solid composition. 39.-46. (canceled)
 47. The method of claim 38, wherein said introducing comprises adding a blowing agent to the cross-linked recombinant resilin solid composition.
 48. The method of claim 47, wherein the blowing agent comprises a chemical blowing agent or a physical blowing agent.
 49. The method of claim 48, wherein the chemical blowing agent comprises sodium bicarbonate, potassium bicarbonate, ammonium, azodicarbonamide, isocyanate, hydrazine, isopropanol, 5-phenyltetrazole, triazole, 4,4′oxybis(benzenesulfonyl hydrazide) (OBSH), trihydrazine triazine (THT), hydrogen phosphate, tartaric acid, citric acid, and toluenesulphonyl semicarbazide (TSS).
 50. (canceled)
 51. (canceled)
 52. The method of claim 48, wherein the physical blowing agent comprises chlorofluorocarbon (CFC), dissolved nitrogen, N₂, CH₄, H₂, CO₂, Ar, pentane, isopentane, hexane, methylene dichloride, and dichlorotetra-fluoroethane.
 53. (canceled)
 54. (canceled)
 55. A composition comprising a cross-linked resilin composition in a polar nonaqueous solvent, wherein said cross-linked resilin is foamed. 56.-58. (canceled)
 59. The method of claim 1, wherein said cross-linked recombinant resilin solid composition comprises a hardness of at least 10 as measured using a Shore 00 Durometer via ASTM D2240.
 60. The method of claim 1, wherein said cross-linked recombinant resilin solid composition comprises a rebound resilience from about 40% to about 60% as measured by ASTM D7121.
 61. The method of claim 1, wherein said cross-linked recombinant resilin solid composition comprises a compressive stress at 25% of about 6 to about 8 psi as measured by ASTM D575.
 62. The method of claim 1, wherein said cross-linked recombinant resilin solid composition does not undergo an elastic to plastic transition below 2 kN of compressive force as measured by a Zwick compression test.
 63. The method of claim 1, wherein said cross-linked recombinant resilin solid composition does not comprise a cross-linking enzyme or a photocatalyst. 